Cell-cycle dependent phosphorylation of yeast pericentrin regulates

1
Cell-cycle dependent phosphorylation of yeast pericentrin regulates -TuSC-
2
mediated microtubule nucleation
3
4
Tien-chen Lin1,3, Annett Neuner1, Yvonne Schlosser1, Annette Scharf2, Lisa Weber2 and
5
Elmar Schiebel1,*
6
1-Zentrum für Molekulare Biologie der Universität Heidelberg (ZMBH), DKFZ-ZMBH Allianz,
7
Im Neuenheimer Feld 282, 69120 Heidelberg, Germany
8
2-Zentrum für Molekulare Biologie der Universität Heidelberg (ZMBH), DKFZ-ZMBH Allianz,
9
Z-MS, Im Neuenheimer Feld 282, 69120 Heidelberg, Germany
10
3-The Hartmut Hoffmann-Berling International Graduate School of Molecular and Cellular
11
Biology (HBIGS), University of Heidelberg, Heidelberg, 69120, Germany
12
13
*Correspondence: [email protected]
14
Key words: pericentrin, Spc110, Spc98, spindle pole body, Mps1, Cdk1, gamma-tubulin
15
complex, phosphorylation, mitosis, microtubules, oligomerization
16
17
Summary
18
Budding yeast Spc110, a member of -tubulin complex receptor family (-TuCR), recruits -
19
tubulin complexes to microtubule (MT) organizing centers (MTOCs). Biochemical studies
20
suggest that Spc110 facilitates higher-order -tubulin complex assembly (Kollman et al.,
21
2010). Nevertheless the molecular basis for this activity and the regulation are unclear.
22
Here we show that Spc110 phosphorylated by Mps1 and Cdk1 activates -TuSC
23
oligomerization and MT nucleation in a cell cycle dependent manner. Interaction between
24
the N-terminus of the -TuSC subunit Spc98 and Spc110 is important for this activity.
25
Besides the conserved CM1 motif in -TuCRs (Sawin et al., 2004), a second motif that we
26
named Spc110/Pcp1 motif (SPM) is also important for MT nucleation. The activating Mps1
27
and Cdk1 sites lie between SPM and CM1 motifs. Most organisms have both SPM-CM1
28
(Spc110/Pcp1/PCNT) and CM1-only (Spc72/Mto1/Cnn/CDK5RAP2/ myomegalin) types of
29
-TuCRs. The two types of -TuCRs contain distinct but conserved C-terminal MTOC
30
targeting domains.
31
32
Highlights
33
34
1. Phosphorylation of Spc110 N-terminal domain (N-Spc110) regulates -TuSC
oligomerization.
35
2. Both SPM and CM1 are required for N-Spc110 to activate -TuSC oligomerization.
36
3. The conserved N-Spc98 is required for Spc110-mediated oligomerization.
37
38
4. -TuCRs can be categorized into CM1-only (Spc72/Mto1/Cnn/CDK5RAP2) and
SPM-CM1 (Spc110/Pcp1/PCNT) type of receptors.
39
40
41
5. While the SPM-CM1 type is targeted via the PACT domain to centrosomes/SPBs,
the CM1-only type has either MASC (Mto1 and Spc72 C-terminus) or a CM2 motif
for MTOC targeting.
42
Introduction
43
The budding yeast spindle consists of ~40 microtubules (MTs) that extend between the two
44
opposed spindle pole bodies (SPBs). Because of the closed mitosis in yeast, the SPBs
45
remain embedded in the nuclear membrane throughout mitosis. Cell-cycle regulated
46
spindle assembly begins in S-phase with nucleation of MTs onto the surface of the newly
47
assembled SPB. As soon as MTs emanate from both SPBs, they interdigitate (pole to pole
48
MTs) or attach to the kinetochores (pole to kinetochore MTs) at the end of S phase, forming
49
the bipolar spindle (O'Toole et al., 1999).
50
The -tubulin complex is the core player in MT nucleation. In budding yeast Saccharomyces
51
cerevisiae, two molecules of -tubulin (Tub4) assemble together with one molecule of
52
Spc97 (ortholog of human GCP2) and Spc98 (ortholog of human GCP3) into a tetrameric -
53
tubulin small complex (-TuSC), which is conserved in all eukaryotes (Geissler et al., 1996;
54
Guillet et al., 2011; Knop et al., 1997). The purified -TuSC of budding yeast is able to self-
55
oligomerize into symmetric ring-like structures under low salt buffer conditions. The
56
diameter and the pitch of the -TuSC ring resemble that of MT cylinder, suggesting that the
57
-TuSC ring functions as a MT nucleation template. However, the in vitro nucleation activity
58
of the -TuSC assemblies remained poor, presumably because of the suboptimal spacing
59
of every second Tub4 within the -TuSC ring that blocks interaction with tubulin in the MT
60
cylinder (Choy et al., 2009; Kollman et al., 2010; Kollman et al., 2008). The concept of-
61
TuSC oligomerization is further supported by in vivo measurements of budding yeast -
62
tubulin complex components on detached single MT nucleation sites. The -
63
tubulin:Spc97:Spc98 ratio was 2.4:1.0:1.3 with a total of ~17 -tubulin molecules per
64
nucleation site (Erlemann et al., 2012), suggesting a slight excess of -tubulin and Spc98
65
molecules over Spc97 in the assembled MT nucleation site.
66
In most eukaryotic organisms, such as fission yeast, Drosophila, Xenopus and human,
67
multiple -TuSCs further assemble with additional GCP family members (GCP4-6) into the
68
larger -tubulin ring complex (-TuRC) (Anders et al., 2006; Gunawardane et al., 2000;
69
Murphy et al., 2001; Zhang et al., 2000). However these additional GCP proteins are not
70
encoded in the budding yeast genome.
71
Various proteins are involved in the recruitment of -tubulin complexes to microtubule
72
organizing centers (MTOCs) such as centrosomes and SPBs. The small protein Mozart1 is
73
encoded in most eukaryotic genomes except the one of budding yeast (Teixido-Travesa et
74
al., 2012). In Schizosaccharomyces pombe and Arabidopsis thaliana Mozart1 interacts with
75
the GCP3 subunit of -tubulin complexes (Batzenschlager et al., 2013; Dhani et al., 2013;
76
Janski et al., 2012; Masuda et al., 2013; Nakamura et al., 2012). In S. pombe Mozart1
77
appears important for the -TuSC recruitment to SPBs (Dhani et al., 2013; Masuda et al.,
78
2013).
79
Besides Mozart1, a group of conserved proteins called -tubulin complex receptors (-
80
TuCRs) are required for targeting -tubulin complexes to MTOCs. Most of them carry a
81
highly conserved centrosomin motif 1 (CM1) that interacts with GCP subunits of -tubulin
82
complexes (Sawin et al., 2004). How Mozart1 and -TuCRs cooperate is not understood.
83
However, in budding yeast cells that lack a Mozart1 gene, -TuCRs are the sole factors
84
responsible for -TuSC recruitment to SPBs. Spc110 is the budding yeast homolog of
85
pericentrin (PCNT) and functions as -TuCRs at the nuclear side of the SPB (Knop and
86
Schiebel, 1997; Sundberg and Davis, 1997). The N-terminal Spc110 encompasses the
87
CM1 that interacts with the Spc98 subunit of -TuSC (Fong et al., 2008; Knop and Schiebel,
88
1997; Nguyen et al., 1998; Samejima et al., 2008; Sawin et al., 2004; Vinh et al., 2002;
89
Zhang and Megraw, 2007). In addition, the N-terminal region of Spc110 is phosphorylated
90
in a cell-cycle dependent manner. Phospho-Spc110 appears as cells progress from S
91
phase, continues accumulating during mitosis and vanishes at the anaphase onset
92
(Friedman et al., 1996; Stirling and Stark, 1996). Spc110 phosphorylation accounts for the
93
impact of Cdk1 and Mps1 kinases on spindle dynamics (Friedman et al., 2001; Huisman et
94
al., 2007; Liang et al., 2013). However, a clear understanding behind this observation is
95
lacking. Interestingly, when -TuSC and an N-terminal fragment of Spc110 (amino acids 1-
96
220 of Spc110; Spc1101-220) were co-expressed in insect cells, a filament-like -TuSC-
97
Spc1101-220 complex formed. The nucleation capacity of this purified -TuSC-Spc1101-220
98
complex exceeded that of the -TuSC alone (Kollman et al., 2010). Thus, Spc1101-220
99
influences -TuSC properties with yet unclear mechanism.
100
Here we have tested the possibility that phosphorylation of the -TuCR Spc110 regulates
101
MT nucleation by inducing -TuSC oligomerization. Single particle analysis of -TuSC
102
incubated with phospho-mimetic Spc110 mutant proteins showed that Mps1 and Cdk1
103
promoted MT nucleation through Spc110 phosphorylation. Phosphorylated Spc110 and the
104
interaction with the N-terminal domain of Spc98 induce -TuSC oligomerization. In addition,
105
bioinformatic analysis revealed a conserved region around T18, that we named
106
Spc110/Pcp1 motif (SPM). SPM and CM1 motifs are both important for -TuSC binding and
107
oligomerization. A comparison of -TuCRs for the presence of SPM and CM1, identified
108
SPM-CM1 (Spc110, Pcp1, PCNT) and CM1-only types of -TuCRs (Spc72, Mto1, Cnn,
109
CDK5RAP2, myomegalin) in most organisms. While the SPM-CM1 type of -TuCRs carries
110
the PACT domain and is targeted only to the centrosome or the nuclear side of the SPB,
111
the CM1-only type of -TuCRs has either a MASC (Mto1 and Spc72 C-terminus) (Samejima
112
et al., 2010) or a CM2 motif and is recruited to, centrosomes, the cytoplasmic side of the
113
SPB or acentrosomal MTOCs.
114
115
Results
116
117
Phosphorylation of N-Spc110 at Mps1 and Cdk1 sites is required for efficient
118
interaction with -TuSC
119
To test whether Spc1101-220 phosphorylation promoted -TuSC ring formation, we purified
120
GST-Spc1101-220 (named Spc1101-220) from both E. coli and the baculovirus expression
121
system. Spc1101-220 encompasses Cdk1 and Mps1 phosphorylation sites and the
122
conserved CM1 motif (Figure 1A). Because of the post-translational modification
123
machinery, Spc1101-220 purified from insect cells harboured phosphorylations on S60/T68
124
and S36/S91 (Figure 1—figure supplement 1A-D), that correspond to established Mps1 and
125
Cdk1 sites, respectively (Figure 1A) (Friedman et al., 2001; Huisman et al., 2007). In
126
contrast, Spc1101-220 was not phosphorylated when purified from E. coli.
127
We incubated purified -TuSC with either the phosphorylated Spc1101-220 from insect cells
128
(Spc1101-220-P) or the non-phosphorylated Spc1101-220 from E. coli and then analyzed the
129
protein complexes by gel filtration. Spc110 dimerizes via the coiled-coli region in the centre
130
of the protein (Kilmartin et al., 1993; Muller et al., 2005). To substitute for the lack of this
131
region, we performed the assays with GST-Spc1101-220 that dimerizes via GST-GST
132
interactions. We used TB150 buffer in our assay instead of the BRB80 buffer used by
133
Kollman et al. (2010) (Kollman et al., 2010). BRB80 induces oligomerization of -TuSC
134
without the need for addition of Spc110 (Figure 1—figure supplement 2A, B). In contrast, in
135
TB150 buffer the majority of -TuSC was monomeric (Figure 1—figure supplement 2C).
136
TB150 therefore allowed us to monitor the impact of Spc1101-220 on -TuSC oligomerization
137
by gel filtration. Addition of Spc1101-220-P shifted -TuSC into fractions that eluted earlier
138
than when -TuSC was run on the columns on its own (Figure 1B, shift from faction 10 to
139
fraction 8). The peak at the void-volume (fraction 8) likely represented -TuSC oligomers. In
140
contrast, non-phosphorylated Spc1101-220 expressed in E. coli did not change the -TuSC
141
elution profile. The -TuSC pool eluted as monomeric -TuSC (Figure 1B, fractions 10-11).
142
This result suggests that Mps1 and Cdk1 kinases regulate the interaction between Spc110
143
and -TuSC through phosphorylation of Spc110 N-terminal domain.
144
145
Phosphorylation of N-Spc110 regulates the -TuSC oligomerization promoting
146
activity
147
To further confirm that phosphorylation of Spc1101-220 alters the interaction with -TuSC,
148
phosphomimetic and non-phosphorylatable Spc1101-220 mutant proteins were purified from
149
E. coli (Figure 2—figure supplement 1A). spc1102A (Cdk1 sites: S36A, S91A) and spc1103A
150
(Mps1 sites: S60A, T64A, T68A) have been studied in vivo before and shown to behave as
151
non-phosphorylatable mutants (Friedman et al., 2001; Huisman et al., 2007; Liang et al.,
152
2013). In addition, we analyzed the phosphomimetic Spc1101-220-2D (Cdk1 sites: S36D and
153
S91D), Spc1101-220-3D (Mps1 sites: S60D, T64D, T68D) and Spc1101-220-5D (Cdk1 and Mps1
154
sites: S36D, S60D, T64D, T68D and S91D). Migration of all purified Spc1101-220 mutant
155
proteins was comparable upon gel filtration, indicating that the mutations did not alter the
156
overall structure of the protein (Figure 2—figure supplement 1B, C).
157
We used gel filtration chromatography to address whether Spc1101-220 mutant proteins
158
induce higher-order -TuSC oligomers. The phosphomimetic Spc1101-220-2D (Cdk1 sites),
159
Spc1101-220-3D (Mps1 sites) and Spc1101-220-5D (Cdk1 and Mps1 sites) induced an apparent
160
shift of -TuSC towards the void-volume compared to the -TuSC only run or -TuSC plus
161
non-phosphorylated Spc1101-220-WT from E. coli (Figure 2A-C and Figure 2—figure
162
supplement 2A, B). The behavior of the phosphomimetic Spc110 mutant proteins reflected
163
that of in vivo phosphorylated Spc1101-220-P purified from insect cells (Figure 1B), indicating
164
that the mutations to acidic residues did mimic phosphorylation. In contrast, the -TuSC
165
shift was not seen upon inclusion of non-phosphorylatable Spc1101-220-5A (Cdk1 and Mps1
166
sites) with the -TuSC (Figure 2—figure supplement 2A, B). Thus, Spc1101-220-5A behaved
167
as the non-phosphorylated Spc1101-220 purified from E. coli, suggesting that the non-
168
phosphorylatable mutations did not impair the protein. This conclusion was further
169
supported by the observation that Spc1101-220-2D or Spc1101-220-3D still possessed
170
oligomerization-promoting activity when combined with non-phosphorylatable mutations
171
(Spc1101-220-2D3A and Spc1101-220-2A3D, Figure 2—figure supplement 3). Taken together, the
172
gel filtration experiment suggests that phosphorylated Spc110 induces oligomerization of -
173
TuSC.
174
Because the upper limit of the separation range of the Superose 6 gel filtration column is
175
~5000 kDa, the -TuSC oligomers in the void-volume contain at least 14 copies of -TuSC
176
(14-mers). This indicates the presence of ring-like complexes or even higher-order
177
oligomeric structures in the void-volume. To confirm the existence of structurally organized
178
oligomers, fractions corresponding to the void-volume peak (Figure 2—figure supplement
179
2A, fraction 7) were subjected to EM analysis (Figure 2D, E and Figure 2—figure
180
supplement 4A). Consistent with the low A280 absorbance, hardly any oligomeric -TuSC
181
species were found in the void fraction of -TuSC only or -TuSC incubated with the non-
182
phosphorylated Spc1101-220-WT (Figure 2E and Figure 2—figure supplement 4B). In
183
contrast, oligomeric -TuSC ring-like assemblies were observed when Spc1101-220-2D,
184
Spc1101-220-3D and Spc1101-220-5D (Figure 2D, E and Figure 2—figure supplement 4A, B)
185
had been added to the -TuSC preparation. Interestingly, the percentage of -TuSC spirals
186
increased steadily with increasing numbers of phospho-mimetic mutations from Spc1101-220-
187
2D
188
the number of rings per field was relatively constant (Figure 2—figure supplement 4C). The
189
outer diameter of the ring-like assemblies was on average around 40 nm (Figure 2—figure
190
supplement 4D). Thus, combined phosphorylation of Spc110 by Cdk1 and Mps1 kinases
191
enhances the oligomerization activity of Spc110 better than phosphorylation by either
192
kinase alone.
, Spc1101-220-3D to Spc1101-220-5D (Figure 2E and Figure 2—figure supplement 4B), while
193
194
The SPM motif is important for Spc110 oligomerization promoting activity
195
An additional phosphorylation of N-Spc110 at T18 has been identified in phosphoproteomic
196
studies (Albuquerque et al., 2008; Keck et al., 2011; Lin et al., 2011). However, its precise
197
function remained unclear (Liang et al., 2013). To elucidate the role of Spc110T18
198
phosphorylation, Spc1101-220-T18D, Spc1101-220-2D-T18D (Cdk1 plus T18), Spc1101-220-3D-T18D
199
(Mps1 plus T18), Spc1101-220-5D-T18D (T18D in addition to Mps1 and Cdk1 sites) and
200
Spc1101-220-5D-T18A proteins were purified (Figure 2—figure supplement 1A) and tested for
201
their -TuSC oligomerization promoting activity. Spc1101-220 species with T18D or T18A
202
failed to induce -TuSC oligomerization even when combined with the activating 5D
203
mutations (Figure 2F and Figure 2—figure supplement 2C). Similar data were obtained with
204
Spc1101-220-5D-T18E and Spc1101-220-5D-T18V (Figure 2-supplement 5A). Moreover, spc110T18D,
205
spc110T18E, spc110T18A and spc110T18V showed identical growth defects at 37ºC (Figure 2-
206
supplement figure 5B). Since the effects of T18D/E and T18A/V mutations were
207
indistinguishable, we cannot attribute these mutations as phosphomimetic or non-
208
phosphorylatable. However, our results strongly suggest that these mutations affect the
209
structure of an important yet unappreciated element around T18 that is important for -
210
TuSC oligomerization. Consistently, hardly any oligomeric structures were observed when
211
-TuSC was incubated with Spc1101-220-5D-T18D (Figure 2G).
212
With the sequence alignments of Spc110 orthologs from yeast to human, we observed a
213
conserved motif upstream of CM1, which we designated as Spc110/Pcp1 motif (SPM)
214
(Figure 2H). Spc110T18 sits within this motif. To evaluate the relative importance of CM1
215
and SPM, we constructed the Spc1101-220-CM1-QA mutant protein with mutations (KE to QA)
216
in two highly conserved residues of the CM1 (Figure 2—figure supplement 6) and the
217
Spc1101-220-ΔSPM lacking the SPM motif (deletion of amino acids 1-20). Spc1101-220 proteins
218
were incubated with -TuSC in TB150 buffer to examine their oligomerization capacity.
219
Spc1101-220-5D-CM1-QA and Spc1101-220-5D-ΔSPM mutant proteins failed to activate -TuSC
220
oligomerization even when the five activating Cdk1 and Mps1 mutations were present
221
(Figure 2I). Thus, Spc1101-220-5D-ΔSPM behaved as Spc1101-220-5D-T18D (compare Figure 2I with
222
Figure 2—figure supplement 2C), further emphasizing that T18D inactivates the SPM.
223
Taken together, SPM and CM1 of Spc110 are both required for -TuSC oligomerization.
224
225
The SPM and CM1 motifs and the Cdk1 and Mps1 phosphorylations regulate the
226
affinity of Spc110 to -TuSC
227
Phosphorylation of Spc110 may regulate the -TuSC oligomerization by changing its
228
binding affinity. Therefore we performed GST-pulldown assay to measure the binding of the
229
Spc1101-220 variants to -TuSC. A constant amount of -TuSC was incubated with
230
increasing concentrations of Spc1101-220 variants (0 to 300 nM). Consistent with the -TuSC
231
oligomerization assay (Figure 2), Spc1101-220-5D showed stronger (p=0.0017 for
232
Spc97/Spc98 and p=0.0006 for Tub4) -TuSC binding than Spc1101-220-WT (Figure 3A, B).
233
Interestingly, while Spc1101-220-2D and Spc1101-220-3D induced comparable levels of -TuSC
234
oligomerzation (Figure 2B), Spc1101-220-2D showed less -TuSC binding than Spc1101-220-3D.
235
A likely explanation is the 30-fold reduction in concentration that was used in the GST-
236
pulldown assay compared to -TuSC oligomerization assays. These results suggested that
237
phosphorylation of Spc110 on S36, S60, T64, T68 and S91 promote -TuSC binding to
238
Spc110.
239
We also analyzed binding of SPM (T18D and ∆SPM) and CM1 (CM1-QA) deficient
240
Spc1101-220 mutant proteins to -TuSC. Both Spc1101-220-5D-T18D and Spc1101-220-5D-CM1-QA
241
exhibited reduced -TuSC binding relative to Spc1101-220-5D (p<0.01 for Spc97/Spc98 and
242
p<0.05 for Tub4) (Figure 3C, D). The -TuSC binding capacity of Spc1101-220-5D-T18D-CM1-QA
243
was further reduced (p<0.01, Figure 3C, D), suggesting both CM1 and SPM are important
244
for -TuSC binding. Interestingly, while Spc1101-220-5D-T18D, Spc1101-220-5D-CM1-QA and
245
Spc1101-220-ΔSPM all failed to oligomerize -TuSC (Figure 2F and 2I), they showed different
246
levels of -TuSC binding capacity (Figure 3D). The decoupling of -TuSC binding capacity
247
from -TuSC oligomerization suggests that both processes are not inevitably linked. Taken
248
together, we conclude that both CM1 and SPM motifs contribute to -TuSC binding and are
249
required for promoting -TuSC oligomerization.
250
251
SPM and the phosphorylation of N-Spc110 control MT nucleation activity in vitro
252
In vitro MT nucleation assays were performed to test the effect of Spc110 variants on -
253
TuSC-mediated MT nucleation. Since phosphorylation of Spc110 and the presence of a
254
functional SPM regulate -TuSC oligomerization into template rings, we expected to see
255
differences in MT nucleation depending on these parameters (Figures 2 and 3). Compared
256
to the buffer control and -TuSC alone, a ~3-fold increase (p<0.05) in MT nucleation activity
257
was observed for Spc1101-220-2D, Spc1101-220-3D or Spc1101-220-5D upon incubation with -
258
TuSC (Figure 4A, B). In contrast, Spc1101-220-WT showed levels of MT nucleation that were
259
comparable to the buffer control and -TuSC alone. Consistent with the proposed role of
260
SPM, the MT nucleation level was reduced to buffer control levels when Spc1101-220-5D-T18D
261
was incubated with -TuSC (p=0.99). These results are in accordance with EM particle
262
quantification assays, as Spc1101-220-2D, Spc1101-220-3D or Spc1101-220-5D induced similar
263
and significantly more -TuSC ring assemblies than Spc1101-220-WT and Spc1101-220-5D-T18D
264
(p<0.05, Figure 2E and Figure 2—figure supplement 4C). In summary, these data support
265
the model that Cdk1 and Mps1 phosphorylations of Spc110 regulate -TuSC-mediated MT
266
nucleation by controlling template assembly.
267
268
Phosphorylation of Spc110 is cell cycle dependent
269
To analyze cell cycle dependent phosphorylation of Spc110, we generated one phospho-
270
specific antibody against the two Cdk1 sites (Spc110S36 and Spc110S91) (Figure 5A) and
271
another against the Mps1 sites on Spc110 (Spc110pS60-pT64-pT68) (Figure 5B). Both anti-
272
Spc110pS36-pS91 and anti-Spc110pS60-pT64-pT68 antibodies gave specific signals in vitro and in
273
vivo (Figure 5A-C).
274
To understand by which Cdk1 kinase complex Spc110S36 and Spc110S91 were
275
phosphorylated, we co-immunoprecipitated Cdk1as1 either in complex with Clb5-TAP (S
276
phase cyclin) or Clb2-TAP (mitotic cyclin) from yeast lysates and performed in vitro kinase
277
assays using Spc1101-220-WT that had been purified from E. coli. Spc110S36-S91 was
278
phosphorylated equally by Cdk1-Clb2 and Cdk1-Clb5 as detected by Spc110pS36-pS91
279
antibodies (Figure 5A). It is important to note that both Cdk1 kinase complexes had equal
280
kinase activities toward histone H3 (Figure 5A, bottom).
281
To confirm the cell-cycle dependent phosphorylation of Spc110 in vivo, we immuno-
282
precipitated (IP) Spc110-GFP with GFP-binder from cells arrested either in G1 phase with
283
-factor, S phase with hydroxyurea (HU) or metaphase with nocodazole, and then blotted
284
with phospho-specific antibodies. At G1 phase, no signal was observed with any of the two
285
phospho-specific antibodies (Figure 5D). The phosphorylation of Mps1 was detected at S
286
phase and maintained at metaphase. The Spc110pS36-pS91 signal also appeared at S phase,
287
and continuously increased at metaphase.
288
T18 within the SPM motif has been found phosphorylated in vivo and matches the minimal
289
Cdk1 consensus sequence S/T-P (Keck et al., 2011). To elucidate the function of T18
290
phosphorylation,
291
phosphorylation assay showed that T18 was only phosphorylated by Cdk1-Clb2 but not by
292
Cdk1-Clb5 (Figure 5—figure supplement 1A). Due to the low sensitivity of anti-Spc110pT18,
293
we used mass spectrometry for the analysis of T18 phosphorylation in vivo. Total yeast cell
294
lysates were fractionated into supernatant and pellet that contained SPBs. The pellet
295
fraction was solublized with high salt buffer and then used for Spc110 enrichment. pT18
296
was predominately detected in the SPB-containing pellet fraction of metaphase cells but not
297
in the corresponding fraction of G1 or S phase arrested cells (Figure 5—figure supplement
298
1B, C). These data suggest that T18 of Spc110 becomes phosphorylated at SPBs in M
we
raised
phosphospecific
antibodies
against
pT18.
In
vitro
299
phase but not S phase, which is consistent with published data (Keck et al., 2011).
300
Finally, we addressed the effects of T18 phosphorylation on -TuSC oligomerization.
301
Because the T18A/V/D/E mutations inactivated the SPM function, we turned to an in vitro
302
phosphorylation approach. Spc1101-220-5D was incubated with Cdk1as1-Clb2 in the presence
303
of absence of the Cdk1as1 inhibitor 1NM-PP1. Only in the absence of 1NM-PP1, Spc1101-
304
220-5D
was phosphorylated on T18 as shown by the phospho-specific anti-T18 antibodies.
305
We incubated Spc1101-220-5D and Spc1101-220-5D-pT18 with -TuSC and analyzed -TuSC by
306
gel filtration. The activity of Spc1101-220-5D-pT18 to promote -TuSC oligomerization was
307
significantly reduced in comparison with Spc1101-220-5D (p<0.05) (Figure 5- figure
308
supplement 1D, E). This suggests that in vitro phosphorylation of T18 by Cdk1-Clb2 inhibits
309
-TuSC oligomerization.
310
Taken together, our results are in agreement with previous reports about the timing of
311
Spc110 phospho-dependent mobility shift in SDS-PAGE (Friedman et al., 1996; Stirling and
312
Stark, 1996) and suggest that the Cdk1 and Mps1 sites become phosphorylated in S and
313
metaphase. In addition, phosphorylation of T18 within SPM occurs at metaphase and may
314
negativly regulate SPM function.
315
316
Cells with spc110 phospho-, SPM- and CM1-mutant alleles have SPBs with impaired
317
MT nucleation activity
318
To reveal the function of phosphorylation of Spc110 in vivo, we replaced SPC110 with
319
phosphomimetic and non-phosphorylatable mutant alleles. There was no significant
320
difference in the expression levels of the Spc110 variants (Figure 6—figure supplement
321
1A). spc1102A/2D, spc1103A/3D, spc1105A/5D, the SPM deficient spc110T18D, spc110∆SPM and
322
the CM1 deficient spc110CM1-QA cells grew at 23ºC comparable to wild-type SPC110 (WT)
323
cells. However, spc110T18D and spc110∆SPM cells displayed a conditional lethal growth
324
defect at 37ºC and the SPM/CM1 defective spc110T18D-CM1-QA double mutant was non-viable
325
at 23ºC and 37ºC (Figure 6A, left panel). Combining T18D with 2D (Cdk1 sites), 3D (Mps1
326
sites) and 5D mutations slightly reduced growth at 37ºC compared to the 2D, 3D and 5D
327
mutations (Figure 6A). This shows that, consistent with our in vitro -TuSC oligomerization
328
results (Figure 2—figure supplement 2C), the SPM mutation T18D is dominant over the
329
activating Cdk1 and Mps1 phosphomimetic mutations.
330
The defect of some mutant alleles involved in spindle assembly becomes apparent only
331
after SAC function has been impaired (Daum et al., 2000; Wang and Burke, 1995). In the
332
absence of the SAC gene MAD2 (Li and Murray, 1991), slow growth was observed in
333
spc1102A and spc1102D cells at 37ºC. This growth defect was even more pronounced in
334
spc1105A mad2Δ cells at 37ºC (Figure 6A, right panel). All spc110 mutant alleles with SPM
335
or CM1 inactivating mutations showed reduced growth in the mad2Δ background compared
336
to MAD2 SAC proficient cells (Figure 6A, bottom panel). Together, these growth tests
337
suggest that the absence of phospho-regulation of Spc110 or the lack of SPM or CM1
338
results in defects that are compensated by a SAC induced delay in cell cycle progression.
339
Based on our model built from in vitro experiments, phosphorylation of N-Spc110 by Cdk1
340
and Mps1 in S phase promotes the binding and oligomerization of -TuSC to form the
341
template required for MT nucleation. In addition, the SPM and the CM1 motifs of Spc110
342
are important for -TuSC oligomerization and MT nucleation (Figure 2). Thus, mutations in
343
SPC110 that affect any one of these sites/motifs should display a reduction in their SPB-
344
associated tubulin signal. To test this hypothesis, we expressed GFP-TUB1 in cells and
345
measured the intensity of the tubulin signal at the SPB at different cell cycle phases. Cells
346
were synchronized with -factor in G1 and then released into pre-warmed media at 37ºC,
347
the restrictive temperature of some of the mutant cells. In SPM defective spc110T18A and
348
spc110T18D cells, the reduction of GFP-tubulin signal at SPBs was already apparent in G1
349
(Figure 6B and Figure 6—figure supplement 1B, p<0.001). In S phase, the stage where
350
newly synthesized SPBs mature and MT nucleation is initiated, all spc110 mutants except
351
for spc1105D cells exhibited a marked reduction in MT nucleation at SPBs when compared
352
with WT cells (Figure 6B; for spc1105A, p<0.05; for spc110T18D, spc110T18A, p<0.001 and
353
Figure 6—figure supplement 1B). The reduction in tubulin signal was especially pronounced
354
in spc110T18D and spc110T18A cells, indicating that SPM mutations blocked MT formation in
355
vivo. spc110∆SPM cells also showed MT defects at 37ºC (Figure 6-supplement 1F).
356
The situation in metaphase was similar to that of S phase although the overall relative
357
differences between WT and spc110 mutants were less pronounced than in S phase
358
(Figure 6B, p<0.001). Consistently, the overall spindle MT signal was reduced in spc1105A,
359
spc110T18A, spc110T18D and spc1105D-T18D cells (Figure 6B, C). Taken together, the in vivo
360
analysis of the tubulin signal at SPBs in spc110 cells is consistent with the in vitro data on
361
-TuSC oligomerization and MT nucleation (Figures 2 and 4).
362
Reduced MT signal intensity at SPBs would be indicative of impaired -TuSC activity. In the
363
simplest interpretation, -TuSC recruitment to SPBs is impaired. We found that the SPB
364
signal from the -TuSC marker Spc97-GFP was reduced in spc1105A, spc110T18D,
365
spc1105D-T18D and spc110∆SPM cells in G1 and S phase compared to SPC110WT and
366
spc1105D cells (Figure 6—figure supplement 1C, G). However, this reduction was not
367
observed in mitotic cells. Analysis of Spc97-GFP signal intensity at SPBs in SPC110 wild
368
type, spc110T18D and spc110∆SPM cells revealed that the SPM mutation allowed Spc97 SPB
369
binding but with a delay in cell cycle timing (Figure 6—figure supplement 1D, G). After SPB
370
separation in late S phase wild type, spc110T18D and spc110∆SPM cells eventually carried
371
similar amounts of Spc97-GFP at SPBs.
372
Analysis of the GFP signal at SPBs in SPC110-GFP, spc1105A-GFP spc110T18D–GFP and
373
spc1105D-T18D-GFP cells did not reveal any differences between cell types (Figure 6—figure
374
supplement 1E). Thus, the reduction of the Spc97-GFP signal in G1 and S phase most
375
likely reflects a reduction of -TuSC binding to the SPC110 mutant molecules at SPBs. The
376
observation that all spc110 cells had similar -TuSC signals at mitotic SPBs while the MT
377
signal was only reduced in spc110T18D and spc1105A cells (Figure 6—figure supplement 1C
378
versus Figure 6B), suggests that the SPB-associated -TuSC is not fully active in MT
379
organization in these cells. The functions of Spc110 probably extend beyond -TuSC
380
binding, most likely to include -TuSC oligomerization (see Discussion). However,
381
compared with spc110T18D or SPC110 cells, Spc110-GFP at the SPB was reduced in
382
spc110∆SPM cells (Figure 6—figure supplement 1H), suggesting the SPM deletion affects
383
incorporation of Spc110 into the SPB. This may contribute to the spc110∆SPM phenotype
384
and may explain why the growth defect of spc110∆SPM cells was more pronounced than that
385
of spc110T18D cells (Figure 6A).
386
To further examine the MT nucleation defect in spc110 phospho-mutant cells, we
387
performed an in vivo MT nucleation assay. Cells were first arrested in S phase with HU,
388
then MTs were depolymerized with nocodazole, followed by nocodazole wash out to trigger
389
MT nucleation by the SPB bound -TuSC (Erlemann et al., 2012). To prevent cell cycle
390
progression into anaphase we retained the cells in the HU block (Figure 6D). The re-growth
391
of MTs was measured as a change in GFP-Tub1 intensity at SPBs. In comparison to WT
392
and spc1105D cells, MT nucleation activity was reduced in the spc1105A, spc110T18A,
393
spc110T18D spc1105D-T18D cells (Figure 6E). Again, these data are consistent with the in vitro
394
data (Figures 1 to 4) and suggest that Spc110’s ability to stimulate MT nucleation is
395
regulated through stimulatory phosphorylation and requires the SPM motif.
396
397
Spc110 interaction and -TuSC oligomerization are facilitated by the N-terminal
398
region of Spc98
399
The N-terminal region of Spc98/GCP3 orthologs is conserved. Spc98/GCP3 family
400
members contain five predicted helices followed by an unstructured linker region (Figure 7A
401
and Figure 7—figure supplement 1A). The conserved GRIP1 and 2 motifs that are common
402
to all GCP proteins follow this N-terminal region (Wiese and Zheng, 2006). Analysis of -
403
TuSC by electron microscopy has shown that the N-terminus of Spc98 is in close proximity
404
to the N-Spc110 binding site (Kollman et al., 2010). This topological arrangement raises the
405
possibility that N-Spc98 is involved in binding to N-Spc110 and oligomerization of -TuSC.
406
To test this idea, we constructed truncations of N-Spc98 with which to assess the function
407
and biochemical behaviour of -TuSCSpc98∆N. All N-terminal truncations of SPC98 supported
408
cell growth at 23ºC (Figure 7B and Figure 7—figure supplement 1B). However, Δ1-156, Δ1-
409
177 and ΔLinker deletions of SPC98 showed reduced or no growth in mad2Δ cells at 37ºC.
410
We analyzed the phenotype of spc98ΔN mutants at their restrictive temperature, 37ºC.
411
Because -TuSCSpc98∆1-177 formed aggregates already in TB150 buffer and was therefore
412
unsuitable for oligomerization assay (Figure 7-supplement figure 2A, B), we focus our
413
analysis on spc98∆1-156. Like the wild-type -TuSC, the purified recombinant-TuSCSpc98∆1was monomeric in TB150 (Figure 7-supplement figure 2C, D). spc98∆1-156 cells
414
156
415
incubated at 37ºC had weaker GFP-Tub1 signal at their SPBs than wild type SPC98 cells
416
(WT, Figure 7C). This is consistent with the observed mild MT nucleation defect of spc98Δ1-
417
156
cells at 37ºC (Figure 7—figure supplement 1C, D). Moreover, time-lapse analysis of -
418
factor synchronized SPC98 SPC42-Cherry and spc98∆1-156 SPC42-mCherry cells with
419
SPC97-GFP or SPC110-GFP revealed a delay in Spc97-GFP recruitment to SPBs at the
420
time of SPB duplication (Figure 7D). Interestingly, slightly more Spc110-GFP was found at
421
SPBs in spc98∆1-156 cells, which may help to compensate the spc98∆1-156 phenotype. Thus,
422
the N-terminal region of Spc98 is important for optimal MT organization and timely -TuSC
423
recruitment to SPBs.
424
We next asked whether -TuSCSpc98∆1-156 can be oligomerized by incubation with Spc1101-
425
. Interestingly, Spc1101-220-5D failed to shift -TuSCSpc98∆1-156 to the void volume of the
220-5D
426
Superose 6 column, as was the case for -TuSC (Figure 7E). In agreement with the
427
oligomerization experiment, -TuSCSpc98∆1-156 showed reduced binding to Spc1101-220-5D in
428
the pull down assay (Figure 7F, G). Thus, the N-terminus of Spc98 is involved in binding to
429
Spc110. In spite of not changing our conclusion, we noticed that Spc98∆1-156-6His was
430
detected less strongly than Spc98-6His by the anti-His antibodies, although in Coomassie
431
Blue stained gels both proteins were present in 1:1 ratio (Figure 7E upper panel and F).
432
Thus, it is likely that Spc98Δ1-156-6His transferred less efficiently to the blotting membrane
433
than Spc97-6His, leading to an underestimation of the Spc98Δ1-156-6His signal relative to
434
full-length Spc98-6His.
435
Finally, we tested -TuSCSpc98∆1-156 oligomerization in the BRB80 buffer that supports
436
oligomerization without the aid of Spc110. -TuSC and -TuSCSpc98∆1-156 shifted to the void
437
volume of the Superose 6 column with equal efficiency (Figure 1—figure supplement 2B
438
and Figure 7 — figure supplement 2C). Analysis of the void fraction by EM identified
439
oligomeric -TuSCSpc98∆1-156 rings (Figure 7—figure supplement 2E). Therefore, the N-
440
terminus of Spc98 is important for Spc110-mediated oligomerization but not for Spc110-
441
independent -TuSC oligomerization.
442
443
Bioinformatic analysis of the C-terminal SPB/centrosomal binding domain of SPM-
444
CM1 and CM1-only -TuCRs
445
Our biochemical and cell biology analysis have identified the novel motif SPM and CM1
446
(Sawin et al., 2004) as important motifs in Spc110 for -TuSC binding and oligomerization.
447
Using bioinformatics approaches, we investigated the presence of SPM and CM1 motifs in
448
-TuCRs from various species (Figure 8A, B). A yet unappreciated CM1 motif was identified
449
in another budding yeast -TuCR, Spc72 (Knop and Schiebel, 1998) (Figure 8A and Figure
450
8—figure supplement 1C).
451
Based on the presence of SPM and CM1 motifs, -TuCRs can be divided into the SPM-
452
CM1 and the CM1-only types. Spc110 and fission yeast Pcp1 fall into the SPM-CM1 class.
453
Our analysis also predicts SPM and CM1 motifs, albeit with moderate sequence deviation,
454
in mammalian PCNT (Figure 8B). Another group of -TuCRs have only a CM1 motif but no
455
SPM. Human CDK2RAP2 and myomegalin, Drosophila Cnn, budding yeast Spc72 and
456
fission yeast Mto1 fall into this class (Figure 8B).
457
A further level of category resolution was achieved by the classification based on the C-
458
terminal MTOC targeting domains (Figure 8—figure supplement 1A, B). It was shown that
459
yeast Spc110 (Stirling and Stark, 2000; Sundberg et al., 1996), Pcp1 (Fong et al., 2010),
460
Drosophila D-PLP (Cp309) (Kawaguchi and Zheng, 2004) and mammalian PCNT
461
(Gillingham and Munro, 2000; Takahashi et al., 2002) are targeted to nuclear side of SPBs
462
or centrosomal MTOCs via the conserved PACT domain (Figure 9A). In case of SPBs that
463
are embedded in the nuclear envelope and therefore have a cytoplasmic and a nuclear
464
side, SPM-CM1 -TuCRs only function in organizing the nuclear MTs that separate the
465
sister chromatids in mitosis (Figure 9B). In contrast, CM1-only -TuCRs of fungi, such as
466
Spc72 in budding yeast (Knop and Schiebel, 1998), Mto1 in fission yeast (Samejima et al.,
467
2010) and ApsB in Aspergillus nidulans (Zekert et al., 2010), have a MASC motif and
468
function at the cytoplasmic side of the SPB or in the cytoplasm. In case of metazoa, CM1-
469
only type of -TuCRs such as CDK5RAP2 (Wang et al., 2010) possess a CM2 domain
470
(Figure 9B and Figure 8—figure supplement 1A), targeting -TuCRs to both centrosomal
471
and acentrosomal MTOCs.
472
In summary, -TuCR family proteins can be classified regarding the -TuSC interaction
473
motifs and the MTOC targeting domain into three groups.
474
475
Discussion
476
Spc110 is a -TuSC-mediated nucleation regulator
477
In this study we show that the -TuCR Spc110 actively participates in MT formation through
478
the SPM and CM1 elements and Cdk1 and Mps1 phosphorylation sites. Binding of -TuSC
479
to Spc110 is a prerequisite for -TuSC oligomerization into the ring-like template that
480
initiates MT assembly. Phosphorylation of Spc110 by Cdk1 and Mps1 regulates -TuSC
481
binding and thereby MT nucleation. Thus, Spc110 is a MT nucleation regulator. We thereby
482
provide a molecular understanding of the previously observed spindle length phenotypes
483
(the two Cdk1-Clb5 sites) and genetic interaction with SPC97 (three Mps1 sites combined
484
with S36) of SPC110 phosphorylation site mutants (Friedman et al., 2001; Huisman et al.,
485
2007). Our data also explain why an N-terminal fragment of Spc110 influences
486
oligomerization of -TuSC when co-expressed in insect cells (Kollman et al., 2010).
487
SPBs duplicate in G1/S phase of the cell cycle in a conservative manner (Adams and
488
Kilmartin, 2000; Byers and Goetsch, 1975; Pereira et al., 2001) (Figure 10A). In late G1, a
489
SPB precursor, named the duplication plaque, assembles on the cytoplasmic side of the
490
nuclear envelope at a specialized substructure of the mother SPB, named bridge (Byers
491
and Goetsch, 1975; Jaspersen and Winey, 2004). In G1/S the duplication plaque then
492
becomes inserted into the nuclear envelope. This allows binding of the Spc110-calmodulin-
493
Spc29 complex from the nuclear side to the central Spc29-Spc42 layer of the embedded
494
duplication plaque (Bullitt et al., 1997; Elliott et al., 1999). In this cell cycle phase Mps1 and
495
Cdk1-Clb5 phosphorylate the N-terminus of Spc110 at five sites to promote -TuSC binding
496
to Spc110 (Figure 10B). Consistently, Cdk1-Clb5 associates with SPBs throughout this time
497
window (Huisman et al., 2007). These phosphorylations increase -TuSC affinity for
498
Spc110-5P and induce -TuSC oligomerization into ring-like complexes that promote MT
499
nucleation (Figure 10B). Phospho-regulation of Spc110 is not absolutely essential for
500
viability as indicated by the growth of spc1105A cells. However, spc1105A cells have growth
501
defects in the absence of the SAC gene MAD2 and fail to organize the full set of MTs in S
502
phase and mitosis (Figure 6). We therefore suggest that Mps1 and Cdk1-Clb5 coordinate
503
the timing of MT nucleation through Spc110 phosphorylation with SPB duplication. A SAC
504
induced cell cycle delay can temper any defects in this phospho-regulation as indicated by
505
the genetic interaction between spc1102A and spc1105A with mad2Δ (Figure 6A).
506
Spc110T18 is an additional Cdk1 in vivo site (Albuquerque et al., 2008; Keck et al., 2011; Lin
507
et al., 2011) (Figure 5—figure supplement 1). Analysis of T18A, T18V, T18E and T18D
508
mutations resulted in similar in vitro and in vivo phenotypes. We have to assume that all
509
these mutations inactivate the important SPM motif in which T18 resides. The fact that T18
510
of Spc110 is phosphorylated only by Cdk1-Clb2 and not by Cdk1-Clb5 and the in vivo
511
phosphorylation of SPB-associated Spc110T18 in mitosis but not in S phase (Keck et al.,
512
2011) (Figure 5—figure supplement 1A-C) indicates that this Cdk1 site is different
513
compared to the two S phase Cdk1 sites S36 and S91 in Spc110. At least in vitro, Cdk1-
514
Clb2 phopshorylation of Spc110T18 inactivates in a dominant manner the -TuSC
515
oligomerization activity (Figure 5—figure supplement 1D, E). Thus, it is well possible that
516
Spc110T18 phosphorylation by Cdk1-Clb2 limits the MT nucleation activity of the SPB
517
associated -TuSC. Most likely only the surplus of -TuSC that is not engaged in MT
518
organization is phosphorylated and affected by Spc110pT18. This model fits with the
519
observation that the yeast SPB of haploid cells organizes a constant number of 20 nuclear
520
MTs during mitosis (Winey et al., 1995).
521
Co-overexpression of SPC97, SPC98 and TUB4 does not induce MT nucleation despite of
522
-TuSC assembly (Pereira et al., 1998). Our and other studies have shown that the missing
523
factor promoting -TuSC ring assembly is Spc110 (Kollman et al., 2010; Vinh et al., 2002).
524
In vivo data indicate that the binding of -TuSC to Spc110, although a prerequisite, is
525
insufficient for MT formation. For example, although the -TuSC component Spc97 bound
526
to mitotic SPBs of spc110T18D or spc1105D-T18D cells with similar efficiency to wild type cells,
527
the -TuSC in the two mutants was insufficient in full MT organization (Figure 6B and Figure
528
6—figure supplement 1C). Consistent with this model, the addition of Spc1101-220-5D-T18D to
529
recombinant -TuSC induced the formation of -TuSC dimers at ~600 kDa (Figure—figure
530
supplement 2A; note that the protein concentration in this experiment was two-times higher
531
than in Figure – figure supplement 2B) without the formation of -TuSC oligomers. We
532
therefore suggest that -TuSC binding to Spc110 and TuSC oligomerization are
533
mechanistically distinct steps (Figure 10C).
534
535
The N-terminus of Spc98 mediates interaction with N-Spc110 and oligomerization
536
The N-terminal region of Spc98 exhibits homology to other GCP3 family members including
537
human GCP3 (hGCP3, Figure 7A and Figure 7—figure supplement 1A). All homologues
538
contain five predicted helical regions that are followed by an unstructured linker region
539
before the two GRIP domains that are common to all GCP proteins (Guillet et al., 2011).
540
Analysis of the structure of yeast -TuSC by electron microscopy localised N-Spc98
541
towards of the base of the Y shaped structure, away from the C-Spc98 that interacts with -
542
tubulin. N-Spc98 is close to the N-terminus of Spc97 and N-Spc110 (Choy et al., 2009;
543
Kollman et al., 2010). This is consistent with yeast two-hybrid studies that identified N-
544
Spc98 as an interactor for Spc110 (Knop and Schiebel, 1997; Nguyen et al., 1998). We
545
have analyzed the importance of N-Spc98 by deleting different portions of this domain
546
including the helices, the linker region and both the helices and the linker. Surprisingly, the
547
N-Spc98 fragment was not essential for the viability at 23ºC (Figure 7B and Figure 7—
548
figure supplement 1B). The important function of this region, however, was revealed at
549
elevated temperatures and when SAC function was impaired (Figure 7B).
550
Biochemical analysis of -TuSCSpc98∆1-156 showed that its binding and ability to be induced
551
to oligomerize by Spc1101-220-5D was reduced (Figure 7E, F and G), although in M phase it
552
bound with the same affinity for SPBs as WT -TuSC (Figure 7D). We suggest that the
553
special SPB arrangement of Spc110 as hexameric units (Muller et al., 2005) compensates
554
in part for the reduced in vito binding of -TuSCSpc98∆1-156 to Spc1101-220. This compensation
555
may arise from cooperative interactions of -TuSC with N-Spc110. Whatever the
556
mechanism, regions of -TuSC in addition to the N-terminal 177 amino acids of Spc98
557
(Figure 7—figure supplement 1B) have to interact with Spc110. Either Spc110 also
558
interacts with the Spc98/GCP3 core or it binds to the N-terminus of Spc97/GCP2.
559
-TuSCSpc98∆1-156 was still able to oligomerize under special buffer conditions (Figure 7—
560
figure supplement 2), which is in agreement with the observation that -TuSCSpc98∆1-156 was
561
functional in vivo and promoted MT nucleation, although defects were seen at elevated
562
temperature and in mad2Δ cells. Thus, the regions that are essential for -TuSC
563
oligomerization await identification.
564
565
-tubulin complex receptors fall into three groups: the SPM-CM1-PACT, CM1-MASC
566
and CM1-CM2 types
567
-TuCRs from yeast to Drosophila to human carry a conserved CM1 motif. This CM1 motif
568
was first identified in fission yeast Mto1 (Sawin et al., 2004). Subsequently the function of
569
CM1 in -TuSC binding was confirmed for Mto1, centrosomin (Drosophila) and CDK5RAP2
570
(human) (Choi et al., 2010; Fong et al., 2008; Samejima et al., 2008; Zhang and Megraw,
571
2007). The -TuCRs Pcp1 (fission yeast) and Spc110 (budding yeast) also contain putative
572
CM1 motifs (Dictenberg et al., 1998; Fong et al., 2010; Takahashi et al., 2002; Zimmerman
573
et al., 2004) (Figure 8B).
574
In addition, we identified a yet unappreciated CM1 motif in the yeast -TuCR Spc72 (Knop
575
and Schiebel, 1998) (Figure 8A). Although the putative CM1 motif in Spc72 appears
576
degenerated compared with the full CM1 of other -TuCRs, the conserved residues shared
577
by Spc72-CM1 and full CM1 suggest that Spc72 contains a functional CM1. Consistently,
578
Spc72 carries a C-terminal MASC, as is the case for other fungal CM1-only -TuCRs
579
(Samejima et al., 2010). Moreover, the predicted CM1 of Spc72 resides in the N-terminal
580
region of Spc72 that is essential for -TuSC binding (Knop and Schiebel, 1998; Usui et al.,
581
2003).
582
Human PCNT falls in terms of structure and function into the -TuCR class (Dictenberg et
583
al., 1998; Doxsey et al., 1994; Gillingham and Munro, 2000). Analysis of the PCNT
584
sequence for CM1 in combination with the newly identified SPM, identified a potential CM1
585
in the N-terminus of PCNT (Figure 8B). This is consistent with the -tubulin binding ability of
586
human PCNT, which lies within the region that contains the SPM-CM1 (Takahashi et al.,
587
2002). In addition, super-resolution analysis of human centrosomes has mapped the
588
localization of -tubulin towards the N-terminus of PCNT (Lawo et al., 2012; Mennella et al.,
589
2012; Sonnen et al., 2012). Experimental evidences are awaited to validate the CM1 motif
590
in Spc72 and PCNT.
591
Our study has identified SPM as a second conserved motif amongst a subgroup of -tubulin
592
complex receptors (Figure 2H), namely the yeast Spc110 homologues, fission yeast Pcp1
593
and human PCNT (Figure 10) (Dictenberg et al., 1998; Doxsey et al., 1994; Flory et al.,
594
2002). The distance between SPM and CM1 is around 90-100 amino acids in the Spc110
595
and Pcp1 type of receptors. In the mammalian PCNT subfamily it is however only around
596
70 amino acids. Whether these differences reflect functionally important differences in
597
regulation or function or are indicative of co-adaptation with alterations in the -TuSC
598
binding sites remains to be seen.
599
The SPM is important for Spc110 binding to -TuSC and induced oligomerization of -TuSC
600
(Figures 3 and 4). Based on these results we propose that SPM co-operates with CM1 to
601
form a bipartite binding element for the -TuSC (Figure 10C). Interestingly, however, other
602
-tubulin complex receptors such as Spc72, fission yeast Mto1, Drosophila Cnn and human
603
CDK5RAP2 carry only the CM1 element but no SPM motif (Figure 8). Strikingly, SPM-CM1
604
type of -TuCRs carry a C-terminal PACT domain that targets these proteins to MTOCs
605
(Figure 8B). The CM1-only type of -TuCRs either contain a MASC (fungi) or a CM2 C-
606
terminal MTOC targeting sequence (metazoa) (Figure 9B). We also noticed that in some
607
metazoa and the fungi phylum Basidiomycota the PACT domain-containing -TuCRs (such
608
as AKAP9 in human and D-PLP in Drosophila) have N-terminal regions distictive from SPM
609
and CM1 motifs, probably reflecting different binding partners in -TuRC. Alltogether, we
610
can classify -TuCRs in at least three classes: CM1-MASC, CM1-CM2 and SPM-CM1-
611
PACT.
612
In budding and fission yeast, the CM1-MASC types of -TuCRs (Spc72 and Mto1) have
613
specific functions in cytoplasmic MT organization, while Spc110 and Pcp1 (SPM-CM1-
614
PACT) organize only nuclear MTs. This functional division is accompanied by the physical
615
separation of the receptors by the nuclear envelope, which remains intact during the closed
616
mitosis of both organisms. Human CDK5RAP2, myomegalin (CM1-CM2 types) and
617
pericentrin (SPM-CM1-PACT type) are associated with the centrosome (Doxsey et al.,
618
1994; Fong et al., 2008). However, CDK5RAP2 and myomegalin also have functions in
619
acentrosomal MT nucleation for example MT nucleation by the Golgi (Choi et al., 2010;
620
Roubin et al., 2013). Thus, it may be the acentrosomal/cytoplasmic function that explains
621
the lack of SPM element in Mto1/CDK5RAP2/myomegalin.
622
Budding yeast organizes MTs only with -TuSC and -TuCRs. Other organisms have
623
additional building blocks that contribute to MT assembly, such as Mozart1 and GCP4-6
624
(Dhani et al., 2013; Masuda et al., 2013). However, GCP4 to 6 are not essential for MT
625
nucleation in fission yeast and Drosophila, while GCP2/Alp4, GCP3/Alp6 and Mozart1 are
626
essential for viability (Anders et al., 2006; Verollet et al., 2006). Puzzlingly, fission yeast
627
Mozart1 is essential for -TuSC recruitment to SPBs (Masuda et al., 2013). This raises the
628
question how budding yeast compensates for Mozart1’s function. GCP3 and -TuCRs of
629
the SPM-CM1 type are conserved between organisms with or without Mozart1. It is likely
630
that small adaptive changes in GCP3 or Spc110 have evolved to compensate for the lack of
631
Mozart1 in budding yeast.
632
What could be the function of -TuCR in organisms that are able to assemble the more
633
stable -TuRC? -TuCRs may recruit and activate the already assembled -TuRC to
634
MTOCs via GCP interactions. In addition, -TuCRs could induce -TuSC assembly into
635
rings that then either function without the help of additional GCPs or subsequently become
636
stabilized by GCP4-6. In any case, considering the conservation between the SPM and
637
CM1 binding elements of -TuRCs and the N-terminal region of Spc98/GCP3, we suggest
638
that the basic principals of MT nucleation are conserved from yeast to human.
639
640
Experimental Procedures
641
642
Plasmid and strain constructions
643
A detailed list of DNA constructs and yeast strains is described in Supplementary File 1 and
644
Supplementary File 2. SPC110 alleles were subcloned into the integration vector pRS304,
645
and SPC98 alleles were subcloned into pRS305K (Christianson et al., 1992; Sikorski and
646
Hieter, 1989; Taxis and Knop, 2006). Point mutations in genes were introduced by PCR-
647
directed mutagenesis and confirmed by DNA sequencing. GST-Spc1101-220 was cloned into
648
pGEX-5X-1 vector (GE Healthcare) and His-tagged Mps1 was cloned into pET28b for
649
expression in BL21 CodonPlus E. coli and into pFastBac1 vector for overexpression in
650
baculovirus-insect cell system. All yeast strains are derivatives of S288c. Gene deletion and
651
epitope tagging of genes at their endogenous loci performed using standard techniques
652
(Janke et al., 2004; Knop et al., 1999). The red fluorescent mCherry was used to mark
653
SPBs through a fusion with SPC42 (Donaldson and Kilmartin, 1996). For SPC97-yeGFP
654
and SPC110-yeGFP strains, the endogenous SPC97 and the integrated SPC110 alleles
655
were tagged with green fluorescent yeGFP. GFP-TUB1 strains were constructed using an
656
integration plasmid (Straight et al., 1997). To remove URA3-based plasmids, transformants
657
were tested for growth on 5-fluoroorotic acid (5-FOA) plates that select against URA3-
658
based plasmids.
659
660
Antibodies
661
Affinity-purified anti-Tub4 (1:1000) and anti-Spc110 (1:1000) antibodies were described
662
previously (Geissler et al., 1996; Spang et al., 1996a; Spang et al., 1996b). Anti-His-tag
663
(1:1000) and anti-GST (1:1000) antibodies were used to detect Spc97 and Spc98 of
664
recombinant -TuSC. Secondary antibodies used in semi-quantitative blotting were
665
IRDye800- or Alexa680-conjugated anti-goat, anti-rabbit, anti-guinea pig and anti-mouse
666
IgGs (1:50,000; Rockland Immunochemicals Inc.). Phospho-specific anti-Spc110pT18, anti-
667
Spc110pS36-pS91 and anti-Spc110pS60-pT64-pT68 antibodies were raised in guinea pigs and
668
purified with immuno-affinity chromatography (1:200; Peptide Specialty Laboratories
669
GmbH).
670
671
Protein purification
672
Subunits of -TuSC and -TuSC∆N-Spc98 were expressed with the baculovirus-insect cell
673
expression system. Purification was performed as described (Gombos et al., 2013).
674
Spc1101-220 variants with an amino-terminal GST tag were purified with Glutathione
675
Sepharose 4B (GE Healthcare) as described (Vinh et al., 2002). Proteins were eluted with 5
676
mM glutathione, concentrated and run over a Superose 6 column equilibrated in HB100 to
677
remove glutathione. Recombinant Mps1 protein was purified in lysis buffer (50 mM
678
NaH2PO4 pH 7.5, 300 mM NaCl, 10% glycerol, 1 mM DTT, 1 mM PMSF, 1X Complete
679
EDTA-free protease inhibitor cocktail (Roche) with column packed with Ni-NTA Sepharose
680
(GE Healthcare). After wash with lysis buffer, protein was eluted with lysis buffer containing
681
200 mM imidazole. Small aliquots for kinase assay were snap frozen with liquid nitrogen
682
and stored at -80°C. Cdk1as1-Clb2 and Cdk1as1-Clb5 complexes were purified from yeast
683
strains kindly given by David Morgan (see Table S1 for detail) (Loog and Morgan, 2005).
684
CLB2-TAP and CLB5-TAP was encoded on 2-micron plasmid and driven by Gal1 promoter.
685
After overexpression with 2% galactose, cells were harvested and lysed for TAP-tag
686
purification as previously described (Ubersax et al., 2003).
687
688
-TuSC oligomerization assay
689
Purified -TuSC and -TuSCSpc98∆N were mixed with or without Spc1101-220 in 1:4 molar ratio
690
(2.3 : 9.3) in TB150 (50 mM Tris-HCl, pH7.0 with 150 mM KCl) or BRB80 (80mM PIPES
691
pH6.9, 1mM EGTA, 1mM MgCl2) buffer as indicated in figure legends. After incubating for 1
692
h at 4°C, the protein mixture was applied to a gel filtration column (Superdex 200 10/300 or
693
Superose 6 10/300 (GE Healthcare)). Elution profiles were recorded as absorbance at 280
694
nm. For each fraction 2% of sample volume was analyzed by SDS-PAGE. Proteins were
695
detected by silver staining or immunoblotting.
696
697
In vitro binding assay
698
For the measurement of the binding of -TuSC to Spc1101-220, recombinant GST (300 nM)
699
or GST- Spc1101-220 proteins (from 0 to 300 nM or fixed 300 nM) were incubated with -
700
TuSC (150 nM) in TB150 buffer in a total reaction volume of 30 μl on a rocking platform for
701
0.5 h at 4°C. Glutathione-Sepharose 4B bead slurry (20 μl; GE Healthcare) was then added
702
to each reaction, followed by rocking for an additional 1 h at 4°C. Beads were washed five
703
times with TB150 buffer, 0.1% NP40 followed by heating in 20 μl SDS sample buffer. Input
704
and bound proteins were analysed by immunoblotting. The signal intensities of protein
705
bands on immunoblots were quantified with ImageJ (NIH). Signal intensities were corrected
706
against the membrane background.
707
708
In vitro MT nucleation assay
709
Microtubule nucleation assay was performed as described (Gombos et al., 2013) with some
710
modifications. 3 μM-TuSC was pre-incubated with 15 μM GST-Spc1101−220 in HB100
711
buffer plus 12.5% glycerol for 30 min on ice. An equal volume of 20 μM bovine brain tubulin
712
containing 4% Alexa568-labelled tubulin in BRB80 buffer with 25% glycerol was added and
713
samples were further incubated for 30 min on ice before being transferred to 37 °°C for
714
15 min for MT polymerization. Samples were fixed with 1% formaldehyde and diluted with 1
715
ml cold BRB80 buffer. 50 l aliquot of reaction mixture was sedimented onto poly-lysine-
716
coated coverslips with a HB-4 rotor at 25,600 g for 1 h. Samples were post-fixed with pre-
717
cooled methanol, mounted on slides with CitiFluor mounting media and imaged with a
718
fluorescence microscope as described. Microtubules were counted in 20 random fields.
719
720
In vitro kinase assay
721
For the in vitro phosphorylation of Spc1101-220 variants, 0.5 g Spc1101-220 substrate was
722
incubated in kinase buffer (50 mM Tris-HCl pH 7.5, 10 mM MgCl2, 2 mM EGTA) with
723
purified Mps1 kinase or Cdk1as1 kinase co-immunoprecipitated with Clb2-TAP or Clb5-TAP.
724
For autoradiography, 5 µCi [-32P]-ATP was added to the reaction mixture and the kinase
725
reaction was incubated for 30 min at 30°C. The reaction was stopped by the addition of
726
SDS sample buffer and analysed by SDS–PAGE, Simply Safe Blue staining (Invitrogen)
727
and autoradiography. For phospho-specific antibody detection, 0.5 mM unlabeled ATP was
728
used in the kinase reaction. For the -TuSC oligomerization assay by gel filtration, the
729
phosphorylation reaction was stopped by addition of 15 M 1NM-PP1 to inhibit Cdk1as1 or
730
10 M SP100625 (Sigma Aldrich) to inhibit Mps1 kinase.
731
732
Electron microscopic analysis of -TuSC oligomers
733
To visualize -TuSC monomers and oligomers, proteins were stained with uranyl acetate
734
and analyzed by electron microscopy. Briefly, we evaporated 300 or 400 mesh
735
copper/palladium grids with a carbon layer on the copper side. To enhance the hydrophilic
736
affinity of the carbon layer, grids were glow discharged for 30~45 sec directly before the
737
preparation started. The protein solution was incubated for 30 sec on the carbon mesh
738
grids at room temperature. The grids were washed and incubated in 2% uranyl acetate for 4
739
min and blotted on Whatman 50 paper. The images were taken in low dose modus with an
740
under-focus between 0.8 to 1.5 m. Particles were viewed with a CM120 electron
741
microscope (Philips Electronics N.V., Eindhoven, Netherlands), which was operated at 120
742
kV. Images were captured with a CCD camera (Keen View, Soft Imaging systems) and
743
viewed with Digital Micrograph Software.
744
Phosphopeptide enrichment and mass spectrometry
745
Phosphopeptides were identified according to Villen et al (Dephoure et al., 2008). In brief,
746
SDS-PAGE separated Spc110 was cut out and subjected to trypsin digestion. After digest,
747
phosphopeptides were enriched by IMAC, starting with 25 µl of PHOS-Select beads
748
(Sigma). Enriched phosphopeptides were eluted and desalted by C18 columns (Stage tips).
749
Peptides were analyzed by LC-MS/MS (Orbitrap Elite, Thermo Fisher Scientific) and data
750
was processed using Thermo proteome discoverer software (1.4). Phosphorylation site
751
localization was performed on the Mascot results using PhosphoRS.
752
753
Growth assay
754
Yeast cells in the early log phase were adjusted to an OD600 of 1 with PBS. Ten-fold serial
755
dilutions of cells were spotted onto the indicated plates and incubated as indicated in the
756
figure legends.
757
758
Quantification of the Spc97-GFP, Spc110-GFP, and GFP-Tub1 signal at SPBs
759
Asynchronous cells were grown in filter-sterilized YPD with additional 0.1 mg/l adenine
760
(YPAD) to an OD600 of 0.3 at 23°C for 3 h and then shifted to 37°C for 1 h. Cells were
761
directly sampled for image acquisition. For the image acquisition, z-stack images with 21
762
0.3 μm steps (2 × 2 binning) were acquired at 37°C with a DeltaVision RT system (Applied
763
Precision) equipped with FITC (fluorescein isothiocyanate), TRITC (tetramethyl rhodamine
764
isothiocyanate) and Cy5 filters (Chroma Technology), a 100x NA 1.4 plan Apo oil
765
immersion objective (IX70; Olympus), and a CCD camera (CoolSNAP HQ; Roper
766
Scientific). Images were processed and analyzed in ImageJ (NIH).
767
The quantification of the mean background intensity and mean fluorescence intensity
768
of Spc97-GFP and Spc110-GFP signals at SPBs was performed on planes having SPBs in
769
focus. Spc97-GFP or Spc110-GFP intensity within the 3×3 pixel-area covering a single SPB
770
or two unsplit SPBs was measured. For GFP-Tub1 at SPBs, GFP-intensity within a 3×3
771
pixel-area surrounding the SPB was measured. Unsplit SPBs were measured together,
772
whereas split SPBs were measured separately. The standard error (SEM) for each data set
773
(n=50) and level of significance was determined by one-way ANOVA with Turkey’s multiple
774
comparisons’ test.
775
776
Time-lapsed live cell imaging
777
Cells were synchronized with -factor for 1.5 cell cycles and then immobilized onto glass-
778
bottom dishes. Dishes were prepared by incubating them with 100 μl concanavalin A
779
solution (6% concanavalin A, 100 mM Tris-Cl, pH 7.0, and 100 mM MnCl2) for 5 min and
780
subsequently washed with 300 μl of distilled water. Yeast cells were allowed to attach to the
781
dishes for 5–15 min at 30°C. The -factor blocked cells were then released by two washes
782
with 2 ml of prewarmed SC medium. Image acquisition was started 10–15 min after release
783
from -factor. Conditions for imaging were as follows: 15 stacks in the FITC channel, 0.1-s
784
exposure, 0.3-μm stack distance, one reference image in bright field channel with a 0.05-s
785
exposure, and 61 frames in total every 3 min. Images were quantified by measuring the
786
integrated density of the sum of projected videos for the region of interest (ROI) around the
787
SPBs and a background region selected from the periphery of the analyzed regions. The
788
mean intensity of the background was subtracted from the ROI. To correct for acquisition,
789
bleaching signal intensities were divided by a bleaching factor. The bleaching factor was
790
determined from the mean of three very short videos that had been generated with the
791
same image acquisition conditions. The data points of bleaching factor were fitted with non-
792
linear one-phase decay equation.
793
794
Nocodazole washout assay (MT regrow assay)
795
Cells (5 × 106 cells/ml) were pre-grown at 23°C in filter-sterilized YPAD. Cells were arrested
796
in G1 by treatment with 10 μg/ml α-factor (Sigma-Aldrich) for 3 h at 23°C until >95% of cells
797
showed a mating projection. G1 arrested cells were released into media with hydroxyurea
798
to arrest cells in S-phase for 2 h. The culture was then shifted to 37°C and nocodazole was
799
added. After 1 h, nocodazole was removed by exchanging media with YPAD containing
800
hydroxyurea. After removal of nocodazole, cells were sampled at 0, 30 and 60 min and
801
fixed with 4% paraformaldehyde. Image acquisition and GFP-Tub1 quantification were
802
performed as described in previous section.
803
804
Trichloroacetic acid (TCA) extraction of yeast cells
805
To measure the expression level of Spc110 variants and GFP-Tub1 in vivo, whole cell
806
lysates were prepared for SDS-PAGE and immunoblotting (Janke et al., 2004; Knop et al.,
807
1999). 2-3 OD600 of late-log phase liquid culture were resuspended in 0.2 M NaOH and
808
incubated on ice for 10 min. 150 μl 55% (w/v) TCA was added, the solutions were mixed
809
and incubated for 10 min on ice. After centrifugation the supernatant was removed. The
810
protein pellet was resuspended in high urea (HU) buffer (8 M urea, 5% SDS, 200 mM Tris-
811
HCl pH 6.8, 0.1 mM EDTA, 100 mM DTT, bromophenol blue) and heated at 65°C for 10
812
min. One-fifth of total sample amount was loaded for SDS-PAGE and western blotting.
813
814
Pull-down of Spc110-GFP with GFP-binder protein
815
For in vivo phospho-Spc110 detection, strains with integrated SPC110-yeGFP or SPC110-
816
TAP alleles were lysed in binding buffer (25 mM HEPES, pH 7.4, 150 mM NaCl, 1 mM
817
EDTA, 1 mM DTT, 50 mM NaF, 80 mM -glycerophosphate, 0.2 mM Na3VO4, and protease
818
inhibitors) lysed with acid-washed glass beads (Sigma-Aldrich) in a FastPrep FP120 Cell
819
Disrupter (Thermo Scientific). Cell lysates were incubated with 0.1% Triton X-100 for 15 min
820
and then clarified by centrifugation at 10,000 g for 10 min. To further solubilize the pellet
821
with insoluble SPBs, binding buffer containg 0.5 M NaCl and 1% Triton X-100 was added to
822
resuspend the pellet and incubated for 40 min. GFP-binder protein (Rothbauer et al., 2008)
823
conjugated Sepharose 4B slurry or IgG-conjugated Dynabeads was added into
824
supernatants and rocked at 4oC for 2 h, followed by five times of washing with binding
825
buffer. Pull-downed Spc110-GFP or Spc110-TAP was eluted by heating with SDS sample
826
buffer and analyzed by SDS-PAGE and immunoblotting or mass spectrometry.
827
828
Bioinformatic analysis
829
Protein sequences of Spc110 and Spc98 and their homologues in selected organisms were
830
aligned with MAFFT algorism built in Jalview software (Katoh and Standley, 2013;
831
Waterhouse et al., 2009) (Figure 2H, 7A, Figure 2—figure supplement 6, Figure 8—figure
832
supplement 1). The threshold of conservation was set as 20% and highlighted. The relative
833
frequency of each amino acid in each position of SPM and CM1 motifs was visualized using
834
the WebLogo 2.0 (Crooks et al., 2004) (Figure 2H, Figure 8, Figure 9, Figure 2—figure
835
supplement 6 and Figure 8—figure supplement 1). To demonstrate the occurrence of SPM
836
and CM1 motifs in selected members of -TuSC receptor family, 47 protein sequences
837
covering the putative CM1 motifs were subjected to MEME motif scanning analysis (Bailey
838
et al., 2009) (Figure 8B).
839
Jpred 3 and Disopred predicted the secondary structure of the N-terminal domain of
840
Spc98 and human GCP3 (Cole et al., 2008; Ward et al., 2004). Domain positions and
841
protein interacting regions of Spc110 and Spc98 (Figure 1A and Figure 7—figure
842
supplement 1A) were illustrated according to -TuSC binding studies of Spc72- and
843
Spc110-truncated forms and to the yeast-two-hybrid studies (Knop and Schiebel, 1998;
844
Sundberg and Davis, 1997; Usui et al., 2003).
845
For the identification of full CM1 motif on Spc110s and degenerative CM1 on Spc72, CM1
846
motif containing -TuCR protein sequences were retrieved from Pfam database
847
(Microtub_assoc, Pfam id: PF07989) and multiple-aligned with Spc110s and Spc72s from
848
species of subphylum Saccharomycotina by MAFFT multiple sequence alignment algorism.
849
To define the CM2 motif pattern, CM2 sequences from human CDK5RAP2 and Drosophila
850
Cnn were used to retrieve protein sequences containing putative CM2 with HMMER server
851
(Finn et al., 2011). The retrieved protein sequences were multiple-aligned with CDK5RAP2
852
and Cnn by MAFFT algorism and the multiple-aligned CM2 sequences were subjected to
853
Weblogo 3.0 server to visualize the pattern.
854
855
Statistical analysis
856
Statistical analysis of fluorescent intensities, immunoblotting intensities and in vitro
857
microtubule numbers was performed with GraphPad Prism 6.1. One-way ANOVA with
858
Turkey’s multiple comparisons’ test was used to compare samples and to obtain adjusted p
859
values. The number of repeated experiments and sample size are indicated in figure
860
legends. No statistical method was used to predetermine sample size. The experiments
861
were not randomized. The investigators were not blinded to allocation during the
862
experiments and outcome assessment. In general the data showed normal distribution.
863
864
Acknowledgements
865
This work was supported by a grant of the Deutsche Forschungsgemeinschaft (Schi295-3-
866
2). Dr. David Morgen is acknowledged for yeast strains. We thank U. Jäkle and S. Heinze
867
for excellent technical support. T.L. is a member of the international graduate school HBIGS
868
and support by a fellowship of the Graduiertenkolleg Regulation of Cell Division.
869
870
References
871
872
873
874
875
876
877
878
879
880
881
882
883
884
885
886
887
888
889
890
891
892
893
894
895
896
897
898
899
900
901
902
903
904
905
906
907
908
909
910
911
912
913
914
915
916
917
Adams, I.R., and Kilmartin, J.V. (1999). Localization of core spindle pole body (SPB) components during SPB
duplication in Saccharomyces cerevisiae. J Cell Biol 145, 809-823.
Adams, I.R., and Kilmartin, J.V. (2000). Spindle pole body duplication: a model for centrosome duplication?
Trends Cell Biol 10, 329-335.
Albuquerque, C.P., Smolka, M.B., Payne, S.H., Bafna, V., Eng, J., and Zhou, H. (2008). A multidimensional
chromatography technology for in-depth phosphoproteome analysis. Mol Cell Proteomics 7, 1389-1396.
Anders, A., Lourenco, P.C., and Sawin, K.E. (2006). Noncore components of the fission yeast gamma-tubulin
complex. Mol Biol Cell 17, 5075-5093.
Bailey, T.L., Boden, M., Buske, F.A., Frith, M., Grant, C.E., Clementi, L., Ren, J., Li, W.W., and Noble, W.S.
(2009). MEME SUITE: tools for motif discovery and searching. Nucleic acids research 37, W202-208.
Batzenschlager, M., Masoud, K., Janski, N., Houlne, G., Herzog, E., Evrard, J.L., Baumberger, N., Erhardt, M.,
Nomine, Y., Kieffer, B., et al. (2013). The GIP gamma-tubulin complex-associated proteins are involved in
nuclear architecture in Arabidopsis thaliana. Front Plant Sci 4, 480.
Bullitt, E., Rout, M.P., Kilmartin, J.V., and Akey, C.W. (1997). The yeast spindle pole body is assembled around
a central crystal of Spc42p. Cell 89, 1077-1086.
Byers, B., and Goetsch, L. (1975). Behavior of spindles and spindle plaques in the cell cycle and conjugation
of Saccharomyces cerevisiae. Journal of bacteriology 124, 511-523.
Choi, Y.K., Liu, P., Sze, S.K., Dai, C., and Qi, R.Z. (2010). CDK5RAP2 stimulates microtubule nucleation by the
gamma-tubulin ring complex. J Cell Biol 191, 1089-1095.
Choy, R.M., Kollman, J.M., Zelter, A., Davis, T.N., and Agard, D.A. (2009). Localization and orientation of the
gamma-Tubulin Small Complex components using protein tags as labels for single particle EM. J Struct Biol.
Christianson, T.W., Sikorski, R.S., Dante, M., Shero, J.H., and Hieter, P. (1992). Multifunctional yeast highcopy-number shuttle vectors. Gene 110, 119-122.
Cole, C., Barber, J.D., and Barton, G.J. (2008). The Jpred 3 secondary structure prediction server. Nucleic
acids research 36, W197-201.
Crooks, G.E., Hon, G., Chandonia, J.M., and Brenner, S.E. (2004). WebLogo: a sequence logo generator.
Genome research 14, 1188-1190.
Daum, J.R., Gomez-Ospina, N., Winey, M., and Burke, D.J. (2000). The spindle checkpoint of Saccharomyces
cerevisiae responds to separable microtubule-dependent events. Curr Biol 10, 1375-1378.
Dephoure, N., Zhou, C., Villen, J., Beausoleil, S.A., Bakalarski, C.E., Elledge, S.J., and Gygi, S.P. (2008). A
quantitative atlas of mitotic phosphorylation. Proceedings of the National Academy of Sciences of the United
States of America 105, 10762-10767.
Dhani, D.K., Goult, B.T., George, G.M., Rogerson, D.T., Bitton, D.A., Miller, C.J., Schwabe, J.W., and Tanaka, K.
(2013). Mzt1/Tam4, a fission yeast MOZART1 homologue, is an essential component of the gamma-tubulin
complex and directly interacts with GCP3(Alp6). Molecular biology of the cell 24, 3337-3349.
Dictenberg, J.B., Zimmerman, W., Sparks, C.A., Young, A., Vidair, C., Zheng, Y., Carrington, W., Fay, F.S., and
Doxsey, S.J. (1998). Pericentrin and gamma-tubulin form a protein complex and are organized into a novel
lattice at the centrosome. J Cell Biol 141, 163-174.
Donaldson, A.D., and Kilmartin, J.V. (1996). Spc42p: a phosphorylated component of the S. cerevisiae spindle
pole body (SPD) with an essential function during SPB duplication. J Cell Biol 132, 887-901.
Doxsey, S.J., Stein, P., Evans, L., Calarco, P.D., and Kirschner, M. (1994). Pericentrin, a highly conserved
centrosome protein involved in microtubule organization. Cell 76, 639-650.
Elliott, S., Knop, M., Schlenstedt, G., and Schiebel, E. (1999). Spc29p is a component of the Spc110p
subcomplex and is essential for spindle pole body duplication. Proc Natl Acad Sci U S A 96, 6205-6210.
Erlemann, S., Neuner, A., Gombos, L., Gibeaux, R., Antony, C., and Schiebel, E. (2012). An extended γ-tubulin
ring functions as a stable platform in microtubule nucleation. J Cell Biol 197, 59-74.
918
919
920
921
922
923
924
925
926
927
928
929
930
931
932
933
934
935
936
937
938
939
940
941
942
943
944
945
946
947
948
949
950
951
952
953
954
955
956
957
958
959
960
961
962
963
964
965
966
967
968
Finn, R.D., Clements, J., and Eddy, S.R. (2011). HMMER web server: interactive sequence similarity searching.
Nucleic acids research 39, W29-37.
Flory, M.R., Morphew, M., Joseph, J.D., Means, A.R., and Davis, T.N. (2002). Pcp1p, an Spc110p-related
calmodulin target at the centrosome of the fission yeast Schizosaccharomyces pombe. Cell Growth Differ 13,
47-58.
Fong, C.S., Sato, M., and Toda, T. (2010). Fission yeast Pcp1 links polo kinase-mediated mitotic entry to
gamma-tubulin-dependent spindle formation. EMBO J 29, 120-130.
Fong, K.W., Choi, Y.K., Rattner, J.B., and Qi, R.Z. (2008). CDK5RAP2 is a pericentriolar protein that functions in
centrosomal attachment of the gamma-tubulin ring complex. Mol Biol Cell 19, 115-125.
Friedman, D.B., Kern, J.W., Huneycutt, B.J., Vinh, D.B., Crawford, D.K., Steiner, E., Scheiltz, D., Yates, J., 3rd,
Resing, K.A., Ahn, N.G., et al. (2001). Yeast Mps1p phosphorylates the spindle pole component Spc110p in
the N-terminal domain. J Biol Chem 276, 17958-17967.
Friedman, D.B., Sundberg, H.A., Huang, E.Y., and Davis, T.N. (1996). The 110-kD spindle pole body
component of Saccharomyces cerevisiae is a phosphoprotein that is modified in a cell cycle-dependent
manner. J Cell Biol 132, 903-914.
Geissler, S., Pereira, G., Spang, A., Knop, M., Souès, S., Kilmartin, J., and Schiebel, E. (1996). The spindle pole
body component Spc98p interacts with the γ-tubulin-like Tub4p of Saccharomyces cerevisiae at the sites of
microtubule attachment. EMBO J 15, 3899-3911.
Gillingham, A.K., and Munro, S. (2000). The PACT domain, a conserved centrosomal targeting motif in the
coiled-coil proteins AKAP450 and pericentrin. EMBO Rep 1, 524-529.
Gombos, L., Neuner, A., Berynskyy, M., Fava, L.L., Wade, R.C., Sachse, C., and Schiebel, E. (2013). GTP
regulates the microtubule nucleation activity of gamma-tubulin. Nat Cell Biol 15, 1317-1327.
Guillet, V., Knibiehler, M., Gregory-Pauron, L., Remy, M.H., Chemin, C., Raynaud-Messina, B., Bon, C.,
Kollman, J.M., Agard, D.A., Merdes, A., et al. (2011). Crystal structure of gamma-tubulin complex protein
GCP4 provides insight into microtubule nucleation. Nat Struct Mol Biol.
Gunawardane, R.N., Martin, O.C., Cao, K., Zhang, L., Dej, K., Iwamatsu, A., and Zheng, Y. (2000).
Characterization and reconstitution of Drosophila gamma-tubulin ring complex subunits. J Cell Biol 151,
1513-1524.
Huisman, S.M., Smeets, M.F., and Segal, M. (2007). Phosphorylation of Spc110p by Cdc28p-Clb5p kinase
contributes to correct spindle morphogenesis in S. cerevisiae. J Cell Sci 120, 435-446.
Janke, C., Magiera, M.M., Rathfelder, N., Taxis, C., Reber, S., Maekawa, H., Moreno-Borchart, A., Doenges,
G., Schwob, E., Schiebel, E., et al. (2004). A versatile toolbox for PCR-based tagging of yeast genes: new
fluorescent proteins, more markers and promoter substitution cassettes. Yeast 21, 947-962.
Janski, N., Masoud, K., Batzenschlager, M., Herzog, E., Evrard, J.L., Houlne, G., Bourge, M., Chaboute, M.E.,
and Schmit, A.C. (2012). The GCP3-interacting proteins GIP1 and GIP2 are required for gamma-tubulin
complex protein localization, spindle integrity, and chromosomal stability. The Plant cell 24, 1171-1187.
Jaspersen, S.L., and Winey, M. (2004). The budding yeast spindle pole body: structure, duplication, and
function. Annu Rev Cell Dev Biol 20, 1-28.
Katoh, K., and Standley, D. (2013). MAFFT multiple sequence alignment software version 7: improvements in
performance and usability. Molecular biology and evolution 30, 772-780.
Kawaguchi, S., and Zheng, Y. (2004). Characterization of a Drosophila centrosome protein CP309 that shares
homology with Kendrin and CG-NAP. Mol Biol Cell 15, 37-45.
Keck, J.M., Jones, M.H., Wong, C.C., Binkley, J., Chen, D., Jaspersen, S.L., Holinger, E.P., Xu, T., Niepel, M.,
Rout, M.P., et al. (2011). A cell cycle phosphoproteome of the yeast centrosome. Science 332, 1557-1561.
Kilmartin, J.V., Dyos, S.L., Kershaw, D., and Finch, J.T. (1993). A spacer protein in the Saccharomyces
cerevisiae spindle poly body whose transcript is cell cycle-regulated. J Cell Biol 123, 1175-1184.
Knop, M., Pereira, G., Geissler, S., Grein, K., and Schiebel, E. (1997). The spindle pole body component
Spc97p interacts with the gamma-tubulin of Saccharomyces cerevisiae and functions in microtubule
organization and spindle pole body duplication. EMBO J 16, 1550-1564.
Knop, M., and Schiebel, E. (1997). Spc98p and Spc97p of the yeast gamma-tubulin complex mediate binding
to the spindle pole body via their interaction with Spc110p. EMBO J 16, 6985-6995.
969
970
971
972
973
974
975
976
977
978
979
980
981
982
983
984
985
986
987
988
989
990
991
992
993
994
995
996
997
998
999
1000
1001
1002
1003
1004
1005
1006
1007
1008
1009
1010
1011
1012
1013
1014
1015
1016
1017
Knop, M., and Schiebel, E. (1998). Receptors determine the cellular localization of a gamma-tubulin complex
and thereby the site of microtubule formation. The EMBO journal 17, 3952-3967.
Knop, M., Siegers, K., Pereira, G., Zachariae, W., Winsor, B., Nasmyth, K., and Schiebel, E. (1999). Epitope
tagging of yeast genes using a PCR-based strategy: more tags and improved practical routines. Yeast 15, 963972.
Kollman, J.M., Polka, J.K., Zelter, A., Davis, T.N., and Agard, D.A. (2010). Microtubule nucleating gamma-TuSC
assembles structures with 13-fold microtubule-like symmetry. Nature.
Kollman, J.M., Zelter, A., Muller, E.G., Fox, B., Rice, L.M., Davis, T.N., and Agard, D.A. (2008). The structure of
the gamma-tubulin small complex: implications of its architecture and flexibility for microtubule nucleation.
Mol Biol Cell 19, 207-215.
Lawo, S., Hasegan, M., Gupta, G., and Pelletier, L. (2012). Subdiffraction imaging of centrosomes reveals
higher-order organizational features of pericentriolar material. Nature cell biology 14, 1148-1158.
Li, R., and Murray, A.W. (1991). Feedback control of mitosis in budding yeast. Cell 66, 519-531.
Liang, F., Richmond, D., and Wang, Y. (2013). Coordination of Chromatid Separation and Spindle Elongation
by Antagonistic Activities of Mitotic and S-Phase CDKs. PLoS Genet 9, e1003319.
Lin, T.C., Gombos, L., Neuner, A., Sebastian, D., Olsen, J.V., Hrle, A., Benda, C., and Schiebel, E. (2011).
Phosphorylation of the yeast gamma-tubulin Tub4 regulates microtubule function. PLoS One 6, e19700.
Loog, M., and Morgan, D.O. (2005). Cyclin specificity in the phosphorylation of cyclin-dependent kinase
substrates. Nature 434, 104-108.
Masuda, H., Mori, R., Yukawa, M., and Toda, T. (2013). Fission yeast MOZART1/Mzt1 is an essential gammatubulin complex component required for complex recruitment to the MTOC, but not its assembly. Molecular
biology of the cell.
Mennella, V., Keszthelyi, B., McDonald, K., Chhun, B., Kan, F., Rogers, G., Huang, B., and Agard, D. (2012).
Subdiffraction-resolution fluorescence microscopy reveals a domain of the centrosome critical for
pericentriolar material organization. Nature cell biology 14, 1159-1168.
Muller, E.G., Snydsman, B.E., Novik, I., Hailey, D.W., Gestaut, D.R., Niemann, C.A., O'Toole, E.T., Giddings,
T.H., Jr., Sundin, B.A., and Davis, T.N. (2005). The organization of the core proteins of the yeast spindle pole
body. Mol Biol Cell 16, 3341-3352.
Murphy, S.M., Preble, A.M., Patel, U.K., O'Connell, K.L., Dias, D.P., Moritz, M., Agard, D., Stults, J.T., and
Stearns, T. (2001). GCP5 and GCP6: two new members of the human gamma-tubulin complex. Mol Biol Cell
12, 3340-3352.
Nakamura, M., Yagi, N., Kato, T., Fujita, S., Kawashima, N., Ehrhardt, D.W., and Hashimoto, T. (2012).
Arabidopsis GCP3-interacting protein 1/MOZART 1 is an integral component of the gamma-tubulincontaining microtubule nucleating complex. Plant J 71, 216-225.
Nguyen, T., Vinh, D.B., Crawford, D.K., and Davis, T.N. (1998). A genetic analysis of interactions with Spc110p
reveals distinct functions of Spc97p and Spc98p, components of the yeast gamma-tubulin complex. Mol Biol
Cell 9, 2201-2216.
O'Toole, E.T., Winey, M., and McIntosh, J.R. (1999). High-voltage electron tomography of spindle pole bodies
and early mitotic spindles in the yeast Saccharomyces cerevisiae. Mol Biol Cell 10, 2017-2031.
Pereira, G., Knop, M., and Schiebel, E. (1998). Spc98p directs the yeast gamma-tubulin complex into the
nucleus and is subject to cell cycle-dependent phosphorylation on the nuclear side of the spindle pole body.
Mol Biol Cell 9, 775-793.
Pereira, G., Tanaka, T.U., Nasmyth, K., and Schiebel, E. (2001). Modes of spindle pole body inheritance and
segregation of the Bfa1p-Bub2p checkpoint protein complex. The EMBO journal 20, 6359-6370.
Rothbauer, U., Zolghadr, K., Muyldermans, S., Schepers, A., Cardoso, M.C., and Leonhardt, H. (2008). A
versatile nanotrap for biochemical and functional studies with fluorescent fusion proteins. Mol Cell
Proteomics 7, 282-289.
Roubin, R., Acquaviva, C., Chevrier, V., Sedjai, F., Zyss, D., Birnbaum, D., and Rosnet, O. (2013). Myomegalin
is necessary for the formation of centrosomal and Golgi-derived microtubules. Biology open 2, 238-250.
1018
1019
1020
1021
1022
1023
1024
1025
1026
1027
1028
1029
1030
1031
1032
1033
1034
1035
1036
1037
1038
1039
1040
1041
1042
1043
1044
1045
1046
1047
1048
1049
1050
1051
1052
1053
1054
1055
1056
1057
1058
1059
1060
1061
1062
1063
1064
1065
1066
1067
1068
Samejima, I., Miller, V.J., Groocock, L.M., and Sawin, K.E. (2008). Two distinct regions of Mto1 are required
for normal microtubule nucleation and efficient association with the gamma-tubulin complex in vivo. J Cell
Sci 121, 3971-3980.
Samejima, I., Miller, V.J., Rincon, S.A., and Sawin, K.E. (2010). Fission yeast mto1 regulates diversity of
cytoplasmic microtubule organizing centers. Curr Biol 20, 1959-1965.
Sawin, K.E., Lourenco, P.C., and Snaith, H.A. (2004). Microtubule nucleation at non-spindle pole body
microtubule-organizing centers requires fission yeast centrosomin-related protein mod20p. Curr Biol 14,
763-775.
Sikorski, R.S., and Hieter, P. (1989). A system of shuttle vectors and yeast host strains designed for efficient
manipulation of DNA in Saccharomyces cerevisiae. Genetics 122, 19-27.
Sonnen, K., Schermelleh, L., Leonhardt, H., and Nigg, E. (2012). 3D-structured illumination microscopy
provides novel insight into architecture of human centrosomes. Biology open 1, 965-976.
Spang, A., Geissler, S., Grein, K., and Schiebel, E. (1996a). gamma-Tubulin-like Tub4p of Saccharomyces
cerevisiae is associated with the spindle pole body substructures that organize microtubules and is required
for mitotic spindle formation. J Cell Biol 134, 429-441.
Spang, A., Grein, K., and Schiebel, E. (1996b). The spacer protein Spc110p targets calmodulin to the central
plaque of the yeast spindle pole body. J Cell Sci 109, 2229-2237.
Stirling, D.A., and Stark, M.J. (1996). The phosphorylation state of the 110 kDa component of the yeast
spindle pole body shows cell cycle dependent regulation. Biochem Biophys Res Commun 222, 236-242.
Stirling, D.A., and Stark, M.J. (2000). Mutations in SPC110, encoding the yeast spindle pole body calmodulinbinding protein, cause defects in cell integrity as well as spindle formation. Biochim Biophys Acta 1499, 85100.
Straight, A.F., Marschall, W.F., Sedat, J.W., and Murray, A.W. (1997). Mitosis in living budding yeast:
anaphase A but no metaphase plate. Science 277, 574-578.
Sundberg, H.A., and Davis, T.N. (1997). A mutational analysis identifies three functional regions of the
spindle pole component Spc110p in Saccharomyces cerevisiae. Mol Biol Cell 8, 2575-2590.
Sundberg, H.A., Goetsch, L., Byers, B., and Davis, T.N. (1996). Role of calmodulin and Spc110p interaction in
the proper assembly of spindle pole body compenents. J Cell Biol 133, 111-124.
Takahashi, M., Yamagiwa, A., Nishimura, T., Mukai, H., and Ono, Y. (2002). Centrosomal proteins CG-NAP
and kendrin provide microtubule nucleation sites by anchoring gamma-tubulin ring complex. Mol Biol Cell
13, 3235-3245.
Taxis, C., and Knop, M. (2006). System of centromeric, episomal, and integrative vectors based on drug
resistance markers for Saccharomyces cerevisiae. Biotechniques 40, 73-78.
Teixido-Travesa, N., Roig, J., and Luders, J. (2012). The where, when and how of microtubule nucleation - one
ring to rule them all. Journal of cell science 125, 4445-4456.
Ubersax, J.A., Woodbury, E.L., Quang, P.N., Paraz, M., Blethrow, J.D., Shah, K., Shokat, K.M., and Morgan,
D.O. (2003). Targets of the cyclin-dependent kinase Cdk1. Nature 425, 859-864.
Usui, T., Maekawa, H., Pereira, G., and Schiebel, E. (2003). The XMAP215 homologue Stu2 at yeast spindle
pole bodies regulates microtubule dynamics and anchorage. EMBO J 22, 4779-4793.
Verollet, C., Colombie, N., Daubon, T., Bourbon, H.M., Wright, M., and Raynaud-Messina, B. (2006).
Drosophila melanogaster gamma-TuRC is dispensable for targeting gamma-tubulin to the centrosome and
microtubule nucleation. J Cell Biol 172, 517-528.
Vinh, D.B., Kern, J.W., Hancock, W.O., Howard, J., and Davis, T.N. (2002). Reconstitution and characterization
of budding yeast gamma-tubulin complex. Mol Biol Cell 13, 1144-1157.
Wang, Y., and Burke, D.J. (1995). Checkpoint genes required to delay cell division in response to nocodazole
respond to impaired kinetochore function in the yeast Saccharomyces cerevisiae. Mol Cell Biol 15, 68386844.
Wang, Z., Wu, T., Shi, L., Zhang, L., Zheng, W., Qu, J.Y., Niu, R., and Qi, R.Z. (2010). Conserved motif of
CDK5RAP2 mediates its localization to centrosomes and the Golgi complex. J Biol Chem 285, 22658-22665.
Ward, J.J., McGuffin, L.J., Bryson, K., Buxton, B.F., and Jones, D.T. (2004). The DISOPRED server for the
prediction of protein disorder. Bioinformatics 20, 2138-2139.
1069
1070
1071
1072
1073
1074
1075
1076
1077
1078
1079
1080
1081
1082
1083
1084
1085
1086
1087
Waterhouse, A.M., Procter, J.B., Martin, D.M., Clamp, M., and Barton, G.J. (2009). Jalview Version 2--a
multiple sequence alignment editor and analysis workbench. Bioinformatics 25, 1189-1191.
Wiese, C., and Zheng, Y. (2006). Microtubule nucleation: gamma-tubulin and beyond. J Cell Sci 119, 41434153.
Winey, M., Mamay, C.L., O'Toole, E.T., Mastronarde, D.N., Giddings, T.H., Jr., McDonald, K.L., and McIntosh,
J.R. (1995). Three-dimensional ultrastructural analysis of the Saccharomyces cerevisiae mitotic spindle. J Cell
Biol 129, 1601-1615.
Zekert, N., Veith, D., and Fischer, R. (2010). Interaction of the Aspergillus nidulans microtubule-organizing
center (MTOC) component ApsB with gamma-tubulin and evidence for a role of a subclass of peroxisomes in
the formation of septal MTOCs. Eukaryotic cell 9, 795-805.
Zhang, J., and Megraw, T.L. (2007). Proper recruitment of gamma-tubulin and D-TACC/Msps to embryonic
Drosophila centrosomes requires Centrosomin Motif 1. Mol Biol Cell 18, 4037-4049.
Zhang, L., Keating, T.J., Wilde, A., Borisy, G.G., and Zheng, Y. (2000). The role of Xgrip210 in gamma-tubulin
ring complex assembly and centrosome recruitment. J Cell Biol 151, 1525-1536.
Zimmerman, W.C., Sillibourne, J., Rosa, J., and Doxsey, S.J. (2004). Mitosis-specific anchoring of gamma
tubulin complexes by pericentrin controls spindle organization and mitotic entry. Mol Biol Cell 15, 36423657.
1088
Figure legends
1089
Figure 1. Phosphorylation of N-Spc110 is required for the -TuSC oligomerization
1090
(A)
1091
sites investigated in this study. These sites are located at the N-terminal domain of Spc110,
1092
which directly interacts with -TuSC (Vinh et al., 2002). The conserved centrosomin motif 1
1093
(CM1) (Sawin et al., 2004; Zhang and Megraw, 2007) is also within the N-terminal domain.
1094
The C-terminal domain of Spc110 is involved in the interaction with SPB central plaque
1095
components Spc29 and Spc42 (Adams and Kilmartin, 1999; Elliott et al., 1999). CBD:
1096
calmodulin binding domain.
1097
(B)
1098
oligomerization. Spc1101-220 was expressed and purified from insect cells (middle panel) or
1099
from E. coli (right panel). Only Spc1101-220 from insect cells carried post-translational
1100
modifications (Figure 1—figure supplement 1). Recombinant -TuSC was incubated with
1101
Spc1101-220 or TB150 buffer on ice. Oligomerization of -TuSC-Spc1101-220 was tested by
1102
gel filtration chromatography using a Superdex 200 10/300 column. Peak fractions of the
1103
chromatograms were analysed by SDS-PAGE and silver staining.
Diagram of Spc110’s functional domain organization and the position of phospho-
Post-translational modifications of Spc1101-220 are required for promoting -TuSC
1104
1105
Figure 2. Mps1 and Cdk1 phosphorylation of N-Spc110 stimulates -TuSC
1106
oligomerization
1107
(A)
1108
in Spc110 used in this study. The indicated Spc1101-220 variants were expressed, purified
1109
from E. coli and then tested for -TuSC oligomerization. WT: wild-type; 2A/D: S36A/D,
Summary of combination of non-phosphorylatable and phospho-mimicking mutations
1110
S91A/D; 3A/D: S60A/D, T64A/D, T68A/D; 5A/D: S36A/D, S91A/D, S60A/D, T64A/D,
1111
T68A/D (see Figure 2—figure supplement 1A for SDS-PAGE of purified Spc1101-220
1112
proteins and Figure 2—figure supplement 1C for gel filtration chromatograms).
1113
(B)
1114
220
1115
separated according to size by gel filtration using a Superose 6 column.
1116
(C)
1117
marks statistical significance at p<0.01. Error bars represent SEM. N=3 to 9 for the number
1118
of experiment performed.
1119
(D)
1120
complexes were analyzed with electron microscopy. Representative ring-like structures of
1121
-TuSC-Spc1101-220-2D, -TuSC-Spc1101-220-3D and -TuSC-Spc1101-220-5D. See Figure 2—
1122
figure supplement 4A for additional EM pictures. Scale bar: 50 nm.
1123
(E)
1124
categorized based on the morphology. N is indicated on the figure for the number of fields
1125
analysed.
1126
(F)
1127
incubated with Spc1101-220-WT, Spc1101-220-5D, Spc1101-220-T18A, Spc1101-220-T18D and
1128
Spc1101-220-5D-T18D as described in (B). Bar graph of area ratio of void-volume peak to total
1129
area of chromatogram was calculated as in (C). ** marks statistical significance at p<0.01.
1130
Error bars represent SEM. N=3 to 9 for the number of experiment performed.
Spc1101-220 phospho-mimicking proteins induced -TuSC oligomerization. Spc1101proteins were incubated with -TuSC in TB150 buffer. The reconstituted complexes were
Bar graph of area ratio of void-volume peak to total area of chromatogram of (B). **
Void-volume peak fractions of (B) were subjected to negative staining and protein
Quantification of (D). Shown is the particle number per field. Particles were
Quantification of void-volume fractions of -TuSC chromatograms. -TuSC was
1131
(G)
Quantification of EM. Void-volume peak fractions of (F) were subjected to negative
1132
staining and protein complexes were analyzed with electron microscopy. Particles were
1133
categorized based on the morphology. Shown is the particle number per field. Note, the
1134
Spc1101-220-WT and Spc1101-220-5D graphs are the same as in (E). N is indicated on the
1135
figure for the number of fields analysed.
1136
(H)
1137
human. Residues are marked according to the ClustalX colour scheme. The occurrence of
1138
each amino acid in each position of CM1 motif is presented with Weblogo 2.0.
1139
(I)
1140
Spc1101-220-5D-∆SPM) were incubated with -TuSC in TB150 buffer. The reconstituted
1141
complexes were separated according to size by gel filtration using a Superose 6 column.
Multiple sequence alignment of SPM element of -complex receptors from yeast to
The indicated Spc1101-220 proteins (Spc1101-220-5D, Spc1101-220-5D-CM1-QA and
1142
1143
Figure 3. Phosphorylation of N-Spc110 regulates the affinity to -TuSC
1144
(A)
1145
Spc97-6His and Spc98-6His) and the GST-tagged Spc1101-220 proteins. The bound
1146
proteins were eluted with sample buffer and separated on SDS-PAGE and analyzed by
1147
immunoblotting with anti-His, anti-GST and anti-Tub4 antibodies. Infrared-dye-labelled
1148
secondary antibodies were applied and detected with Li-cor imaging system.
1149
(B)
1150
marks statistical significance at p<0.01. Error bars represent SEM. N=3 for the number of
1151
experiment performed.
GST pull-down assays were performed between -TuSC (containing His-tagged
Quantification for bound Spc97, Spc98 and Tub4 from (A) at 300 nM Spc1101-220. **
Mutations in SPM or CM1 affect -TuSC binding. As (A) but performed with the
1152
(C)
1153
indicated SPM and CM1 mutant Spc110 1-220 constructs.
1154
(D)
1155
300 nM Spc1101-220. ** marks statistical significance at p<0.01 and *** at p<0.001. Error
1156
bars represent SEM. N=3 for the number of experiment performed.
Quantification of Spc1101-220 variants and bound Spc97, Spc98 and Tub4 from (C) at
1157
1158
Figure 4. Spc1101-220 phosphorylation enhances MT nucleation activity in vitro
1159
(A)
1160
phosphorylation. Representative image fields of Alexa546-labeled microtubules from the
1161
nucleation assay polymerized in the presence of buffer, -TuSC and Spc1101-220 variants
1162
(see (B)). Scale bar: 10 µm.
1163
(B)
1164
was counted from 20 fields. The MTs/field was normalized with MTs/field obtained by the
1165
MT nucleation reaction in the presence of buffer only. N=6 to 9 for the number of
1166
independent experiment performed. * marks statistical significance at p<0.05 and ** at
1167
p<0.01. Error bars represent SEM.
Enhancement of MT nucleation activity of Spc110 by Mps1 and Cdk1
Quantification of the MT nucleation assay. For each experiment, the number of MTs
1168
1169
Figure 5. Cell cycle dependent phosphorylation of Spc110
1170
(A, B) Two phospho-specific antibodies were generated from guinea pigs to recognize
1171
phosphorylation of Cdk1 sites (pS36-pS91) (A) and Mps1 sites (p60-p64-pT68) (B). In vitro
1172
kinase assays were performed in the presence of recombinant Mps1 or Cdk1as1 and
1173
Spc1101-220, either wild type (WT) or non-phosphorylatable variants as indicated. 3A (Cdk1)
1174
indicates T18A-S36A-S91A and 3A (Mps1) S60A-T64A-T68A. In vitro phosphorylated
1175
Spc1101-220 was subjected to SDS-PAGE and immunoblot with the corresponding phospho-
1176
specific antibodies. The specific kinase activity of Cdk1as1-Clb2 and Cdk1as1-Clb5 was
1177
compared using human histone H1 as substrate (A, bottom). The numbers in the histone
1178
H1 experiment represent the relative kinase activity. As negative control of kinase activity,
1179
Mps1 was inactivated with chemical inhibitor SP100625 (B), while Cdk1as1-Clb2 and
1180
Cdk1as1-Clb5 overexpressed and purified from budding yeast were inactivated with 1NM-
1181
PP1 (A).
1182
(C)
1183
and spc1105A-GFP cells were arrested in mitosis with nocodazole. Spc110-GFP was
1184
enriched using GFP binder conjugated to beads, and the bound proteins were subject to
1185
immunoblotting with the indicated antibodies.
1186
(D)
1187
Spc110-GFP was enriched with GFP-binder and analyzed by immunoblotting with the
1188
indicated P-specific antibodies. A non-specific band was used as loading control.
Phosphorylation of Spc110 and Spc1105A in vivo. SPC110-GFP wild type cells (WT)
SPC110-GFP wild type cells were arrested in G1, S phase and mitosis as indicated.
1189
1190
Figure 6. Cells with spc110 phospho-, SPM- or CM1-mutant alleles have defects in
1191
spindle formation
1192
(A)
1193
encoding SPC110 (WT) or SPC110 mutants with and without the SAC gene MAD2. Growth
1194
was tested either on synthetic complete (SC) plates containing 5-FOA or SC dropout plates.
1195
(B)
1196
SPC42-mCherry were incubated at 37ºC, the restrictive temperature of some of the spc110
1197
mutants (Figure 6A). The fluorescent signal of GFP-Tub1 at SPBs was quantified in G1
Growth of ten-fold serial dilutions of SPC110 shuffle strains with integration vector
The indicated SPC110 wild type cells and spc110 mutant cells carrying GFP-TUB1
1198
phase cells (no buds), early S phase cells (small bud with unsplit SPBs) and M phase cells
1199
(large bud with split SPBs and spindle length within 2 m). 50 cells were analyzed per cell
1200
cycle phase and strain. Error bars represent SEM. * marks statistical significance at p<0.05,
1201
** at p<0.01, and *** at p<0.001. N=2 or 3 for the number of experiment performed.
1202
(C)
1203
GFP-Tub1 and the Spc42-mCherry signal along the spindle axis. Scale bar: 4.5 µm.
1204
(D)
1205
spc110 cells with GFP-TUB1 were treated as indicated in the outline. The GFP-Tub1 signal
1206
at SPBs was measured at 0, 30 and 60 min after nocodazole washout. N=40 for the
1207
number of cells analyzed per time point and strain. Error bars represent SEM.
1208
(E)
1209
significance at p<0.001.
The representative metaphase cells from (B) were scanned for the distribution of the
In vivo MT re-nucleation assay. Wild-type SPC110 cells (WT) and the indicated
Data of (E) after 30 and 60 min. Error bars represent SEM. *** marks statistical
1210
1211
Figure 7. The N-terminus of Spc98 mediates binding to N-Spc110
1212
(A)
1213
Shown are the putative -helical regions H1-H5. Residues are marked according to the
1214
ClustalX colour scheme.
1215
(B)
1216
37ºC.
1217
(C)
1218
was performed as in Figure 6B. ** marks statistical significance at p<0.01. N=100 cells
1219
analysed for each strain.
Alignment of the amino acid sequence of GCP3 homologues from yeast to human.
Growth test of spc98∆1-156 cells with and without the SAC gene MAD2 at 23ºC and
GFP-Tub1 signal at SPBs in SPC98 wild type or spc98∆1-156 cells. The experiment
SPC98 and spc98∆1-156 cells with SPC42-mCherry, SPC97-GFP or SPC110-GFP
1220
(D)
1221
were analyzed by time lapse analysis. The fluorescent intensity was measured over time
1222
(right). Error bars represent SEM. N=14 and 19 for the number of cells analysed in SPC97-
1223
GFP and SPC-110-GFP strains respectively. The Spc97-GFP and Spc110-GFP signals of
1224
selected cells are shown on the left panel. Scale bar: 5 m.
1225
(E)
1226
buffer. The experiment was performed as in Figure 2B. The purified -TuSCSpc98∆1-156 after
1227
SDS-PAGE and Coomassie Blue staining is shown with relative intensity of the protein
1228
bands. The protein distribution in the Superdex 200 10/300 chromatogram was analyzed
1229
with SDS-PAGE and silver staining.
1230
(F)
1231
reaction was performed and analyzed by immunoblotting as described in Figure 3A.
1232
(G)
1233
Spc1101-220-5D. Error bars represent SEM. N=3 for the number of experiment performed. ***
1234
marks statistical significance at p<0.001.
Spc1105D combined with -TuSCSpc98∆1-156 fails to induce oligomerization in TB150
Binding of -TuSC and -TuSCSpc98∆1-156 to Spc1101-220 mutant proteins. The binding
Quantification of Figure 7F. Pull-downed Spc97, Spc98 and Tub4 proteins at 300 nM
1235
1236
Figure 8. Two types of -TuCRs defined by the N-terminal -TuSC binding motifs:
1237
SPM-CM1 and CM1-only
1238
(A)
1239
alignment of -TuCR protein sequences. The CM1 motif sequence logos were shown for -
1240
TuCR protein sequences retrieved from Pfam database (Microtub_assoc, Pfam id:
1241
PF07989), Spc110s and Spc72s from subphylum Saccharomycotina. The Spc72
1242
sequences used to generate the short CM1 logo are shown in Figure 8—figure supplement
Graphical representations of the patterns of CM1 motif within the multiple sequence
1243
1C. The sequence logos were generated with Weblogo 3.0 server. The conserved residues
1244
shared by CM1 defined by Pfam, Spc110s and Spc72s are marked with asterisks.
1245
(B)
1246
CDK5RAP2/Cnn/Spc72/Mto1 (CM1 type). The occurrences of the motif in the training set
1247
sequences was calculated and shown as coloured blocks on a line with MEME motif
1248
scanning analysis (Bailey et al., 2009). Weblogo motif diagram is shown for each sequence
1249
showing the sites contributing to that motif in that sequence. The best p-value for the
1250
sequence/motif combination is listed. The p-value of an occurrence is the probability of a
1251
single random subsequence with the length of the motif, generated according to the zero-
1252
order background model, having a score at least as high as the score of the occurrence.
SPM and CM1 in PCNT, Spc110 and Pcp1 homologues (SPM-CM1 type) and in
1253
1254
Figure 9. -TuCRs are classified into three subgroups based on N-terminal -TuSC
1255
binding motifs and C-terminal MTOC targeting motifs
1256
(A)
1257
the multiple sequence alignment of -TuCR protein sequences. The MASC and PACT motif
1258
sequence logos were shown for -TuCR protein sequences retrieved from Pfam database
1259
(Mto2_bdg, Pfam id: PF12808; PACT_coil_coil, Pfam id: PF10495) and Spc110s from
1260
subphylum Saccharomycotina. The -TuCR and Spc110 protein sequences used to
1261
generate the CM2 and PACT motif logo are shown in Figure 8—figure supplement 1A, B.
1262
The sequence logos were generated with Weblogo 3.0 server. The conserved residues
1263
shared by PACT motif defined by Pfam and Spc110s are marked with asterisks.
1264
(B)
1265
N-Terminal -TuSC interacting motifs (CM1 and SPM) and C-terminal MTOC targeting
1266
motifs (MASC, CM2 and PACT), -TuCR protein family can be divided into three
Graphical representations of the patterns of C-terminal MTOC targeting motifs within
Summary table of categorization of -TuCR protein family. Based on the presence of
1267
subgroups: N-terminal CM1 motif only with either C-terminal MASC or CM2 motifs, and N-
1268
terminal CM1 and SPM motifs combined with C-terminal PACT domain. Some PACT
1269
domain proteins (AKAP9 or D-PLP) have yet undefined -TuSC binding motifs.
1270
1271
Figure 10. Role of Spc110 phosphorylation during SPB duplication
1272
(A)
MT nucleation by the SPB during the cell cycle. See Discussion for details.
1273
(B)
Cell cycle dependent, stimulatory phosphorylations of Spc110 by Mps1 and Cdk1-
1274
Clb5 (early S phase to early M).
1275
(C)
1276
SPM and CM1 motifs with the N-terminus of Spc98 (GCP3) and possibly also with other
1277
regions of -TuSC.
1278
Figure 1 – figure supplement 1
1279
Phosphorylation of Spc1101-220 from insect cells
1280
(A)
1281
baculovirus-insect cell expression system.
1282
(B)
Mass spectra of identified Mps1 sites (S60 and T68).
1283
(C)
Mass spectra of identified Cdk1 sites (S36 and S91).
1284
(D)
Comparison of investigated phospho-sites of Spc110 N-terminal domain with published data
1285
(Albuquerque et al., 2008; Friedman et al., 2001; Huisman et al., 2007; Keck et al., 2011; Lin et al.,
1286
2011).
Model for the interaction of -TuSC with Spc110. The Spc110 dimer interacts via the
Table of mass-spectrometry identified phosphopeptides of Spc1101-220 purified from
1287
1288
Figure 1 – figure supplement 2
1289
-TuSC does not oligomerize in TB150 buffer
1290
(A-C) Purified -TuSC (A) spontaneously oligomerizes in BRB80 buffer (B) but not in TB150 buffer
1291
(C). Proteins were analyzed by Superose 6 10/300 and subsequently gel filtration fractions were
1292
subjected to SDS-PAGE and silver staining. Samples in the peak fraction were subjected to
1293
negative staining and protein complexes were analyzed by electron microscopy. Corresponding
1294
scale bars are shown on the images.
1295
1296
Figure 2 - figure supplement 1
1297
Purification of Spc1101-200 variants
1298
(A)
1299
PAGE was stained with Coomassie Blue.
1300
(B)
1301
thyroglobulin (667 kDa), ferritin (440 kDa), aldolase (158 kDa), conalbumin (75 kDa), ovalbumin (44
1302
kDa). Partition coefficient (Kav) was calculated using the equation: (Ve-V0)/(Vc-V0), where V0 =
1303
column void volume, Ve = elution volume, and Vc =geometric column volume. The calibration curve
1304
(bottom) of Kav versus log molecular weight was plotted in semi-logarithmic scale.
1305
(C)
1306
TB150 buffer by Superose 6 10/300 chromatography.
Purified GST-Spc1101-220 variants. ~1 g of protein was loaded to each lane. The SDS-
Calibration of Superose 6 10/300 gel filtration column. Following protein markers were used:
Gel filtration chromatograms of purified GST-Spc1101-220 variants. Proteins were analyzed in
1307
1308
Figure 2 – figure supplement 2
1309
Phosphomimetic but not SPM defective N-Spc110 proteins induce oligomerization of
1310
-TuSC
1311
(A)
1312
void-volume (V0, boxed area) corresponds to molecular weight fractions higher than 5000 kDa.
1313
Note, that the concentrations of -TuSC and Spc1101-220 variants in (A) was double as high
Overlapped gel filtration chromatograms with molecular weight markers. The peak of the
1314
compared to (B) and (C).
1315
(B)
1316
phosphorylatable Spc1101-220 mutant proteins. -TuSC was incubated in TB150 buffer with Spc1101-
1317
220
1318
chromatography. Fractions corresponding to the peak were analyzed by SDS-PAGE and silver
1319
staining.
1320
(C)
Gel filtration chromatograms of -TuSC incubated with phosphomimetic or non-
proteins from Figure 2—figure supplement 1A. Complexes were analyzed by Superose 6 10/300
As (B) but with Spc1101-220 variants that have defective SPM due to T18D or T18A.
1321
1322
Figure 2 – figure supplement 3
1323
Non-phosphorylatable mutations in Mps1 or Cdk1 sites of Spc110 are neutral to -TuSC
1324
oligomerization induced by phosphomimetic mutations
1325
Gel filtration chromatograms of -TuSC incubated with indicated Spc1101-220 variants in TB150
1326
buffer. Non-phosphorylatable mutations in addition to phosphomimetic mutations were introduced
1327
on Spc1101-220 to examine the possibility that non-phosphorylatable mutations alter overall structure
1328
and thus disrupt the activity to induce -TuSC oligomerization. Complexes were analyzed by
1329
Superose 6 10/300 chromatography.
1330
1331
Figure 2 – figure supplement 4
1332
EM single particle analysis of oligomerized -TuSC
1333
(A)
1334
50 nm.
1335
(B)
1336
variants. Both the absolute particle number and the percentage of each particle category are shown.
1337
(C)
Additional examples of ring-like and filament-like -TuSC-Spc1101-220 complexes. Scale bar:
Summary of particle numbers of -TuSC oligomerized by Spc1101-220 phospho-mimicking
Spc1101-220-2D, Spc1101-220-3D and Spc1101-220-5D induced similar level of ring-like -TuSC-
1338
Spc1101-220 complexes. * marks statistical significance at p<0.05. Error bars represent SEM. N=15-
1339
25 for the number of field analyzed. Correlated to Figure 2E, G.
1340
(D)
1341
Spc1101-220 phosphomimatic variants. Error bars represent SEM. N=9, 12, 18 for the number of ring-
1342
like complexes measured in 2D, 3D and 5D, respectively.
The diameter of the ring-like -TuSC-Spc1101-220 complexes showed no difference among
1343
1344
Figure 2 – figure supplement 5
1345
Mutations of T18 abolish the -TuSC oligomerization promoting activity by inactivating the
1346
SPM motif.
1347
(A)
1348
additional T18 mutations. Complexes were analyzed by Superose 6 10/300 chromatography in
1349
TB150 buffer.
1350
(B)
1351
vector (null) or SPC110 (WT) or SPC110 mutant alleles on the integration vector. Wild type and
1352
SAC deficient mad2∆ cells were analyzed. Growth was tested either on synthetic complete (SC)
1353
plates containing 5-FOA or SC dropout plates.
Gel filtration chromatograms of -TuSC incubated with phosphomimetic Spc1101-220-5D with
Growth of ten-fold serial dilutions of SPC110 shuffle strains carrying the empty integration
1354
1355
Figure 2 – figure supplement 6
1356
Multiple sequence alignment of CM1 motif-containing proteins
1357
Multiple sequence alignment of selected -TuSC receptor family members containing CM1 motifs.
1358
Two of the most conserved residues within CM1 motif are marked with asterisks and mutated to
1359
disrupt CM1 function in the spc110CM1-QA mutant. The occurrence of each amino acid in each
1360
position of CM1 motif is presented with Weblogo 2.0.
1361
Figure 5 – figure supplement 1
1362
Phosphorylation of T18 most likely affects -TuSC oligomerization promoting activity
1363
of Spc110 in a negative manner
1364
(J)
1365
vitro kinase assays were performed in the presence of Cdk1as1 and Spc1101-220, either wild type
1366
(WT) or non-phosphorylatable Spc1101-220-3A
1367
Spc1101-220 was subjected to SDS-PAGE and immunoblot with the phospho-specific antibody. As
1368
negative control of kinase activity, Cdk1as1-Clb2 and Cdk1as1-Clb5 overexpressed and purified from
1369
budding yeast were inactivated with 1NM-PP1. Note, the same Spc1101-220 samples were analyzed
1370
in Figure 5A with the anti-pS36-pS91 antibody. The Spc1101-220 blot on the bottom is the same as in
1371
Figure 5A.
1372
(K)
1373
data independent fashion in a pseudo MRM experiment in the Orbitrap mass spectrometer
1374
fragmenting peptides with m/z = 689.81. Extracted ion chromatogram of phosphorylated
1375
NMEFTPVGFIK was quantified. Expected retention time of the tryptic phospho-T18 peptide is 24.7
1376
min.
1377
(L)
1378
fragmented ions.
1379
(M)
1380
the presence (+1NM-PP1) and absence of the Cdk1as1 inhibitor 1NM-PP1 and ATP. This allowed
1381
Spc110T18 phosphorylation in the absence but not in the presence of 1NM-PP1. Adding 1NM-PP1
1382
stopped all Cdk1as1 kinase reactions. The so modified Spc1101-220-5D-pT18 was then incubated with -
1383
TuSC. -TuSC was analysed by gel filtration using a Superose 6 column for oligomerization. The gel
1384
filtration fractions were analyzed by immunoblotting for Spc97/Spc98, Tub4, Spc1101-220 and
Phospho-specific antibody against phospho-T18 (pT18) was generated from guinea pigs. In
(Cdk1)
(T18A-S36A-S91A). In vitro phosphorylated
Quantification of tryptic phospho-T18 peptide (NMEFpTPVGFIK). Data was acquired in a
Mass spectrum of fragmented phospho-T18 peptide. The graph summarizes the identified
Gel filtration chromatograms of -TuSC. Spc1101-220-5D was incubated with Cdk1as1-Clb2 in
1385
Spc1101-220-pT18 distribution. The anti-pT18 blot on the bottom is a 25-times of contrast enhancement
1386
of the blot on top. Fractions covering the void-volume peak were marked with a red rectangle
1387
outline.
1388
(N)
1389
control. Indicated protein band intensities of fractions covering the void-volume peak (red rectangle
1390
outline in (D)) were summed and divided by the total intensity of fractions. * marks statistical
1391
significance at p<0.05, ** at p<0.01, and *** at p<0.001. Error bars represent SEM. N=4 for the
1392
number of experiment performed.
Quantification of void-volume fractions shown in (D). Spc1101-220-5D-T18D was included as
1393
1394
Figure 6 – figure supplement 1
1395
Phenotype of SPC110 mutants
1396
(A)
1397
cells grown to OD600 0.5 were harvested and subjected to TCA extraction for whole cell protein
1398
lysates. SDS-PAGE and immunoblotting with anti-Spc110 were performed. Tub2 was loading
1399
control.
1400
(B)
1401
phase (small bud). Microtubules were marked with GFP-Tub1 and SPBs were marked with Spc42-
1402
mCherry. See Figure 6B for experimental set up. BF: bright field. Scale bar: 4.5 µm.
1403
(C)
1404
stages were determined according to cell morphology and spindle length. Unfixed cells were
1405
analyzed for Spc97-GFP signal at SPBs. N = 30 for the number of cells analyzed for each category.
1406
* marks statistical significance at p<0.05 and ** at p<0.01. Error bars represent SEM.
1407
(D)
1408
fluorescent intensity was recorded and measured over time (top). Error bars represent SEM. N=17
There was no significant difference in the protein levels of Spc110 variants. Asynchronous
The representative images of wild type SPC110 (WT) and spc110 cells in G1 (no bud) and S
The indicated wild type SPC110 (WT) and spc110 cells were grown at 37ºC. The cell cycle
SPC110 and spc110T18D cells with SPC97-GFP were analyzed by time lapse analysis. The
1409
for the number of cells analysed for each strains. The Spc97-GFP signals of selected cells are
1410
shown on the bottom. Scale bar: 5 m.
1411
(E)
1412
stages were determined according to cell morphology and spindle length. Unfixed cells were
1413
analyzed for Spc110-GFP signal at SPBs. Error bars represent SEM. N = 30 for the number of cells
1414
analyzed for each category.
1415
(F)
1416
spc110∆SPM carrying GFP-TUB1 SPC42-mCherry were incubated at 37ºC. The fluorescent signal of
1417
GFP-Tub1 at SPBs was quantified in G1 phase cells (no buds), early S phase cells (small bud with
1418
unsplit SPBs) and M phase cells (large bud with split SPBs and spindle length within 2 m). 100
1419
cells were analyzed per cell cycle phase and strain. Error bars represent SEM. ** marks statistical
1420
significance at p<0.01, and *** at p<0.001.
1421
(G)
1422
spc110∆SPM cells with SPC97-GFP were analyzed by time lapse analysis. Error bars represent SEM.
1423
N=17 for the number of cells analysed for each strains.
1424
(H)
1425
were grown at 37ºC. The cell cycle stages were determined according to cell morphology and
1426
spindle length. Unfixed cells were analyzed for Spc110-GFP signal at SPBs. Error bars represent
1427
SEM. N = 40 for the number of cells analyzed for each category.
The indicated wild type SPC110 (WT) and spc110 cells were grown at 37ºC. The cell cycle
Similar experiment as performed in Figure 6B. The indicated SPC110 wild type and
Similar experiment as performed in (D) with SPC110 and spc110∆SPM cells. SPC110 and
Similar experiment as performed in (E). The wild type SPC110 (WT) and spc110SPM cells
1428
1429
Figure 7 – figure supplement 1
1430
The importance of the N-terminal region of Spc98
1431
(A)
1432
Both Spc98 and GCP3 share the conserved N-terminal domain (NTD), the GCP core body and the
1433
divergent, unstructured linker connecting NTD and GCP core body. The GRIP1 region is within the
Structure and functional domain organization of budding yeast Spc98 and human GCP3.
1434
region interacting with Spc97, while the GRIP2 covers the region interacting with Tub4 (Knop et al.,
1435
1997; Kollman et al., 2010; Pereira et al., 1998; Wiese and Zheng, 2006).
1436
(B)
1437
Serial diluted cells were spotted on YPAD plates and incubated at indicated temperatures for two or
1438
three days. SPC98 indicates the spc98∆ SPC98 cells while WT is the unmodified wild type strain.
1439
The design of each truncated variants is shown in the cartoon below.
1440
(C)
1441
within 2 m long). SPC98 wild type and spc98∆1-156 cells with SPC42-mCherry GFP-TUB1 were
1442
grown at 37ºC for 2 h. Metaphase-like cells were analyzed for the presence of bipolar spindles. BF:
1443
bright field. Scale bar: 4 m.
1444
(D)
1445
156
1446
“immature/defective spindle”.
The growth test of MAD2 or mad2∆ cells with spc98 N-terminal truncated mutant alleles.
The representative images of spc98∆1-156 cells in M phase (large budded, bipolar spindle
Quantification of MT phenotype of metaphase cells of (C). One hundred SPC98 and spc98∆1-
cells with a large bud and a SPB distance of ~2 µm were categorized into “normal spindle” or
1447
1448
Figure 7 – figure supplement 2
1449
-TuSCSpc98∆1-156 maintains self-oligomerization capability in BRB80 buffer
1450
(A)
1451
by Superose 6 10/300. The left panel presents the Coomassie Blue stained SDS-gel of purified -
1452
TuSCSpc98∆1-177.
1453
(B)
1454
electron microscopy. The particles showed irregular morphology, suggesting the formation of
1455
protein aggregates. Corresponding scale bars are shown in the images.
1456
(C)
1457
BRB80 buffer and then analyzed by Superose 6 10/300.-TuSCSpc98∆1-156 oligomerized in BRB80
1458
but not in TB150 buffer.
-TuSCSpc98∆1-177 was purified from insect cells, incubated in TB150 buffer and then analyzed
-TuSCSpc98∆1-177 in fraction 7 from (A) were subjected to negative staining and analyzed with
Similar experiment as performed in (A). Purified -TuSCSpc98∆1-156 was incubated in TB150 or
1459
(D, E) Similar experiment as performed in (B). After the gel filtration shown in (C), the peak fraction
1460
of-TuSCSpc98∆1-156 (fraction 7 in BRB80 and fraction 13 in TB150) was subjected to negative
1461
staining and analyzed with electron microscopy. Corresponding scale bars are shown on the
1462
images.
1463

1464
Figure 8 – figure supplement 1
1465
Raw sequence input for the sequence logo of CM2, Spc110 PACT and Spc72 CM1 motifs
1466
(A)
1467
motif. Residues are marked according to the ClustalX colour scheme. The occurrence of each
1468
amino acid in each position of CM2 motif is presented with Weblogo 3.0. The CM2 motif is present
1469
in human CDK5RAP2, myomegalin and Drosophila centrosomin (Cnn).
1470
(B)
1471
Saccharomycotina. The graphic presentation was generated as in (A).
1472
(C)
1473
Saccharomycotina. The graphic presentation was generated as in (A).
Multiple sequence alignment of selected -TuSC receptor family members containing CM2
Multiple sequence alignment of PACT motif sequences of Spc110s from the subphylum
Multiple sequence alignment of CM1 motif sequences of Spc72s from the subphylum
1474
1475
Supplementary File 1
1476
Plasmids used in this study
1477
1478
Supplementary File 2
1479
Strains used in this study
Figure 1- Lin et al
A
T18
Spc29 BD
S36 S91
CBD
CM1
SPM
Coiled-coil
1
183
773
S60 T64 T68
B
Spc42 BD
J-TuSC only
kDa
7
944
Spc1101-220 (E. coli)
Spc1101-220-P (insect cell)
kDa
8 9 10 11 12 13 ml
7
8 9 10 11 12 13 ml
kDa
100
100
100
55
55
55
A280 (mAU)
A280 (mAU)
15
10
5
0
5
7
9
11 13
Elution volumn (ml)
Superdex 200
15
A280 (mAU)
15
20
10
5
0
5
7
9
11 13 15
Elution volumn (ml)
Superdex 200
8 9 10 11 12 13 ml
7
Spc97
Spc98
Tub4
Spc1101-220
25
20
15
10
5
0
5
7
9
11
13
Elution volumn (ml)
Superdex 200
15
Figure 2 - Lin et al
B
**
Percentage of void-volume
peak area to total area
60
20
10
2
1
(ml)
volume
Elution
00
0
se 6 1 /3
Supero
E
D
J-TuSC/Spc1101-220-5D
6
5
4
3
2
1
0
Particles/field
J-TuSC/Spc1101-220-2D
J-TuSC/Spc1101-220-3D
18
14 16
10 12
6 V0 8
F
Oligomer
Ring
Incomplete ring
Spiral
J-TuSC WT
only
N=
Spc1101-220
20
**
2D
3D
5D
15
25
17
22
8D
3
le
-T
1
4
mp
8D
5
5D
6
Sa
8A
0
T1
1.J-TuSC
2.J-TuSC + Spc1101-220-WT
3.J-TuSC + Spc1101-220-2D
4.J-TuSC + Spc1101-220-3D
5.J-TuSC + Spc1101-220-5D
6.J-TuSC + Spc1101-220-5A
30
T
S60 T64 T68
3A/3D
40
W
183 220
Percentage of void-volume
peak area to total area
1
A280 (mAU)
50
5A/5D
5D
CM1
SPM
C
Gel filtration chromatograms
2A/2D
S36 S91
T1
A
Spc1101-220
EFTP G
Ring
Spiral
L
QVN
K
S
R
E
V
K
I
E
Q
R
N
13
12
10
8
11
N
H
Q
N
Q
Q
S
S
A
R
L
L
9
L
E
S
Y I
FQ
J-TuSC + Spc1101-220-5D
C
SPM
Oligomer
Incomplete ring
2
F
0M
N
I
A
7
L
Y
6
1
4
2
5
3
3
4
bits
H
1
6
5
4
3
2
1
0
20
PCP1_SCHPO
A280 (mAU)
Particles/field
G
G8ZV09_TORDC
SP110_ZYGRC
SP110_LACTC
SP110_ASHGO
I6NDW4_ERECY
SPC110_KLULA
SPC110_CANGA
G8BX70_TETPH
SP110_VANPO
SP110_YEAST
15
10
5
0
SP110_SACAR
5
H2AW02_KAZAF
WT
5D
5D-T18D
15
22
16
7
11
13
15
17
Elution volumn (ml)
Superose 6
K0KVJ5_WICCF
G5C7V2_HETGA
PCNT_MOUSE
PCNT_RABIT
PCNT_HUMAN
PCNT_PONAB
PCNT_PANTR
J-TuSC + Spc1101-220-5D-CM1-QA
F6S0J0_MACMU
PCNT_CANFA
A280 (mAU)
20
15
10
5
0
5
7
9
11
13
15
17
Elution volumn (ml)
Superose 6
J-TuSC + Spc1101-220-5D-'SPM
20
A280 (mAU)
N=
9
J7RX36_KAZNA
15
10
5
0
5
7
9
11
13
15
Elution volumn (ml)
Superose 6
-
17
Figure 3 - Lin et al.
C
Spc1101-220-5D
His (Spc97)
His (Spc98)
100
55
Tub4
55
GST (Spc1101-220)
55
His (Spc97)
His (Spc98)
Tub4
55
GST (Spc1101-220)
100
Spc1101-220 (300 nM)
1 2 3 4 5 6
kDa
100
B
Spc97, Spc98
Tub4
**
**
His (Spc97/Spc98)
55
Tub4
55
GST (Spc110-N)
100
His (Spc97/Spc98)
Tub4
55
55
Spc110 1-220
(300 nM)
GST (Spc110-N)
Spc110 1-220
(300 nM)
D
Spc97/Spc98
GST-Spc1101-220
Tub4
***
***
PM
1 QA
M
-'
S
5D
1 QA
-C
-C
M
8D
-T
1
1 QA
-T
1
8D
-
CM
1 QA
5D
-'
SP
M
8D
-T
1
-C
M
5D
5D
5D
5D
PM
1 QA
-'
S
M
-C
8D
-T
1
5D
1 QA
8D
-T
1
-C
M
5D
5D
5D
0
5D
20
8D
40
-T
1
60
5D
80
5D
100
Relative intensity (%)
120
**
***
Relative intensity (%)
140
5D
Relative intensity (%)
P
5D
S
1. GST
2. Spc1101-220-5D
3. Spc1101-220-5D-T18D
4. Spc1101-220-5D-CM1-QA
5. Spc1101-220-5D-T18D-CM1-QA
6. Spc1101-220-5D-'SPM
GST
kDa
Spc1101-220-3D
GST
Spc111-2201-220-2D
GST
Spc1101-220-WT
GST
A
Figure 4 - Lin et al
A
1
2
3
4
5
6
7
8
B
*
*
**
**
J-TuSC Spc1101-220 -
+
-
1
2
-
+
+
+
+
5D WT 2D
3D
5D
5D-T18D
6
7
3
+
**
**
4
5
8
Figure 5 - Lin et al
B
Cdk1as1
Spc1101-220
1NM-PP1
Clb5-TAP
Spc1101-220
W
T
3A
(C
W dk1
T
)
3A
(C
W dk1
T
)
3A
(
W Cdk
T 1)
3A
(C
dk
1)
Clb2-TAP
-
-
+ +
Mps1
-
-
+
W
T
3A
(M
W ps1
T
)
3A
(M
ps
1)
A
-
SP100625
(Mps1 inhib.)
+
55
pS36-pS91
55
Spc1101-220
-
+ +
55
pS60-pT64
-pT68
55
Spc1101-220
kDa
kDa
Cdk1as1
Clb2-TAP Clb5-TAP
1NM-PP1
-
+
-
+
32
P
Histone H1
CBB
1
C
WT
D-factor HU
5A
kDa
pS60-pT64-pT68
(Mps1)
130
pS36-pS91
(Cdk1)
130
Spc110
IP with GFP binder
130
WT
D
Noco
kDa
IP with GFP binder
0.94
(G1)
(S)
Noco
(M)
130
pS60-pT64-pT68
(Mps1)
130
pS36-pS91
(Cdk1)
Loading control
70
Figure 5 - figure supplement 1 - Lin et al
B
A
Retention time (min): 22.98-27.01
Clb2-TAP
Intensity (%)
Cdk1as1
Clb5-TAP
G1, supernatant
100
RT: 24.72
AA: 3075
50
-
-
+ +
-
-
+
Intensity (%)
Intensity (%)
1NM-PP1
+
55
S, supernatant
100
50
0
Intensity (%)
Spc110
1-220
W
T
3A
(C
W dk1
T
)
3A
(C
W dk1
T
)
3A
(
W Cdk
T 1)
3A
(C
dk
1)
0
pT18
100
M, supernatant
RT: 24.76
AA: 6601
50
.
.
.
0
.
100
50
G1, pellet
0
55
Intensity (%)
100
Spc1101-220
Intensity (%)
kDa
S, pellet
50
0
RT: 24.74
AA: 7737
100
M, pellet
50
RT: 25.37
AA: 749
0
23.0
23.5
24.0
24.5
25.0
Time (min)
25.5
26.0
26.5
27.0
C
#1
bƈ
1
b²ƈ
115.05021
Seq.
58.02874
yƈ
y²ƈ
#2
10
N
11
2
262.08562
131.54645 M-Oxidation
1264.56841
632.78784
3
391.12822
196.06775
1117.53299
559.27013
E
9
4
538.19664
269.60196
F
988.49039
494.74883
8
5
719.21065
360.10896
T-Phospho
841.42197
421.21462
7
6
816.26342
408.63535
P
660.40796
330.70762
6
7
915.33184
458.16956
V
563.35519
282.18123
5
8
972.35331
486.68029
G
464.28677
232.64702
4
F
407.26530
204.13629
3
I
260.19688
130.60208
2
K
147.11281
74.06004
1
9
1119.42173
560.21450
10
1232.50580
616.75654
11
E
Tub4
Spc97/Spc98
15
5
17
9
11
13
15
17
Spc1101-220
Elution volume (ml)
Superose 6
Elution volume (ml)
Superose 6
kDa
7
6 7 8 9 10 11 12 13 14 15 1617 ml kDa
100
100
55
55
55
55
55
55
55
55
-
5
PP1
-
Spc1101-220
6 7 8 9 10 11 12 13 14 15 16 17 ml
Spc1101-220
His (Spc97)
His (Spc98)
Tub4
GST (Spc110
pT18
pT18 (contrast
enchanced)
)
20
15
10
5
PP1
1-220
Spc110
***
***
25
1-220-5D
+
5D
13
-
-
+
-
5
5D D
-T
18
D
11
+
5D
9
-
15
5D
7
5
PP1
0
0
15
**
25
5D
5
25
Percentage of total
specified protein (%)
5
Percentage of total
specified protein (%)
10
10
**
35
8D
15
15
**
35
5D
A280 (mAU)
A280 (mAU)
45
20
-T
1
Cdk1 -Clb2 + 1NM-PP1
Percentage of total
specified protein (%)
Cdk1 -Clb2
20
5
*
as1
5D
as1
-
-
5D
-T
18
D
D
-
Figure 6 - Lin et al.
B
A
37 C
800
600
D
5D
5D
WT
null
T18D
CM1QA
T18D-CM1QA
'SPM
18
8A
5D
0
5D
200
0
T1
8D
400
200
5A
Intensity (RU)
400
W
T
5A (Cdk1 +Mps1 sites)
5D (Cdk1 +Mps1 sites)
600
5A
Intensity (RU)
3D (Mps1 sites)
***
1000
800
-T
***
1000
**
T1
23 C
WT
null
2A (Cdk1 sites)
2D (Cdk1 sites)
3A (Mps1 sites)
W
T
23 C
37 C
***
*
o
D
23 C
o
18
23 C
o
o
T1
8D
o
S: GFP-Tub1
-T
o
G1: GFP-Tub1
5-FOA
SC-Trp
5-FOA
T1
8A
SC-Trp
M: GFP-Tub1
***
*** *
Intensity (RU)
1000
WT
null
T18D
2D-T18D
800
600
400
200
3D-T18D
5D
-T
18
D
T1
8D
5D
5A
W
T
T1
8A
0
5D-T18D
MAD2
C
mad2'
M phase
Spc42GFPmCherry Tub1
Spc42GFPmCherry Tub1
BF
WT
4.5 Pm
uorescent intensity
(RU)
1500
uorescent intensity
(RU)
BF
M phase
1000
T18D
500
0
0
1
2
3
4
Distance (Pm)
5A
uorescent intensity
(RU)
uorescent intensity
(RU)
1500
1000
5D-T18D
500
0
0
1
2
3
4
1500
1000
500
0
0
uorescent intensity
(RU)
2
3
4
Distance (Pm)
1500
1000
500
0
0
1
2
3
4
Distance (Pm)
Distance (Pm)
5D
1
1500
1000
500
0
0
1
2
3
4
Distance (Pm)
37oC
30 min
60 min
1 hr
***
60 min
50
0
0
30
60
Time after releasing from nocodazole (min)
Spc1101-220
18
-T
5D
5D
0
D
-50
T1
8D
0
-50
100
T1
8A
50
5D
100
150
5A
***
***
200
W
T
Fluorescent Intensity (RU)
***
W
T
30
***
150
D
60
200
18
WT
5A
5D
T18A
T18D
5D-T18D
***
-T
90
30
T1
8D
0
5D
2 hr
HU
T1
8A
3 hr
E
HU
Noco
HU
5A
D-factor
Fluorescent Intensity (RU)
23oC
Fluorescent Intensity (RU)
D
Spc1101-220
Figure 7 - Lin et al.
A
H2
H1
B
H3
GCP3_ARATH
GCP3_Danio_rerio
23oC
GCP3_CRIGR
GCP3_HUMAN
GCP3_Gallus_gallus
GCP3_XENLA
37oC
YPH499
GCP3_CULQU
GCP3_AEDAE
GCP3_CAMFO
MAD2
GCP3_HARSA
GCP3_ACREC
SPC98
GCP3_SCHPO
GCP3_DROME
spc98'1-156
SPC98_YEAST
H4
H5
YPH499
GCP3_ARATH
GCP3_Danio_rerio
GCP3_CRIGR
mad2'
GCP3_HUMAN
GCP3_Gallus_gallus
SPC98
GCP3_XENLA
spc98'1-156
GCP3_CULQU
GCP3_AEDAE
GCP3_CAMFO
GCP3_HARSA
GCP3_ACREC
GCP3_SCHPO
GCP3_DROME
SPC98_YEAST
D
M: GFP-Tub1
5 Pm
SPC98
**
500
-27
300
-9
-18
200
0
9
18
Spc110-GFP
Abs intensity (RU)
-1
56
T
'1
W
spc98'1-156
-18
0
-9
9
18
-30
-20
-10
0
Time (min)
10
20
30
500
5 Pm
SPC98
-27
50
0
-40
27
100
0
WT: Spc97-GFP
WT: Spc42-mCherry
spc98'1-156: Spc97-GFP
spc98'1-156: Spc42-mCherry
150
100
spc98'1-156
400
Intensity (RU)
200
Spc97-GFP
Abs intensity (RU)
C
27
WT: Spc110-GFP
WT: Spc42-mCherry
spc98'1-156: Spc110-GFP
spc98'1-156: Spc42-mCherry
400
300
200
100
0
-40
-30
-20
-10
0
10
20
30
Time (min)
Spc97 (0.9)
70
55
Spc98'1-156 (0.9)
J-TuSC + Spc1101-220-5D
J-TuSCSpc98'1-156 + Spc1101-220-5D
15
10
1. Spc1101-220-5D
2. GST
3. Spc1101-220-5D
4. Spc1101-220-5D-T18D
5. Spc1101-220-5D-CM1-QA
6. Spc1101-220-5D-T18D-CM1-QA
7. Spc1101-220-5D-'SPM
5
Tub4 (1)
0
6
8
10
12
14
Elution volumn (ml)
Superdex 200
J-TuSC
kDa
100
7
8
9
J-TuSC
kDa
100
10 11 12 13
7
8
9
Spc98'1-156
J-TuSCSpc98'1-156
kDa 1 2 3 4 5
100
55
55
6 7
His (Spc97)
His (Spc98'1-156)
Tub4
GST (Spc1101-220)
10 11 12 13 ml
100
Spc97
Spc98
'1-156
Tub4
Spc1101-220-5D
55
55
C
F
20
J-T
uS
100
Input
Protein (RI)
Resin-bound
kDa
A280 (mAU)
25
E
55
His (Spc97)
His (Spc98'1-156)
Tub4
55
GST (Spc1101-220)
Spc97/Spc98
Tub4
100
Relative intensity (%)
Relative intensity (%)
G
80
60
40
20
0
100
80
60
40
20
0
1
J-TuSC
3
4
5
6
J-TuSCSpc98-'1-156
7
1
J-TuSC
3
4
5
6
J-TuSCSpc98-'1-156
7
Figure 8 - Lin et al.
A
GCP receptor N-terminal CM1 motifs
3.0
2.0
1.0
0.0
L
M
QE
TLKE
RD S
S
RGM
Q
NY
N
V
A
D
ST
LN
Q
MS
E
bits
P
0.0
RQS
I
L
SA
V
G
Q
K
L
AND T
L
E
SQ
0.0
ND
5
V
DRF
D
K
A
A
Q
D
H
K
I
T
G
N
F
V
S
10
S
ST
*** **
Q
NR
L
EIV
RV
A
H
15
C
S
G
H
I
M
K
R
LQ
20
V
Y
IK
L
S
V
25
N ELK
V
EEM
AIKE
LS
AI
KD
Y
G
I
S
V
F SQ
P
N
D
Y
R
Q
V
H
L
S
T
R
I
T
T
R
Q
D
Q
QL
R
K
E
Q
45
ELQ
LQR
KDN
VEK
ET
S
TDL AK
MK
S
R
A
N
V
VL V
G
S
I
T
Q
50
V
K
S
Q
D
R
L
E
T
P
T
M VE
G
I
Q
S
V
35
A
K
E
I
N
S
T
M
N
E
D DD
QR
G
E
L
N L
K
L
E
R
S
F
H
A
E
N
LA
K
P
N
G
N
S
N
S
M
Y
A
A
T
E
A
K
G
EL
R
I
S
L
EQ
G
A
S
S
I
T
45
N
M
Q
F
V
50
SA
I
K
Y
T
M
L
I
Q
F
R
Q
Y
Y
I
L
WK
E
K
A
T
Q
K
S
V
S
PCNT
PCNT_HUMAN
G5C7V2_HETGA
PCNT_MOUSE
G3H8X7_CRIGR
SPC110_YEAST
SP110_SACAR
G0VH34_NAUCC
G8BX70_TETPH
Spc110
G8ZV09_TORDC
SPC110_CANGA
SP110_VANPO
SP110_LACTC
I6NDW4_ERECY
SP110_ASHGO
SPC110_KLULA
SPC110_CANGA
J7RX36_KAZNA
K0KVJ5_WICCF
Q7S473_NEUCR
Q874Y4_PODAS
Pcp1
G2XH36_VERDV
G0SB24_CHATD
C8VIR1_EMENI
Q2UQD3_ASPOR
Q0U842_PHANO
H9FD39_MACMU
PCP1_SCHPO
SPC72_YEAST
MTO1_SCHPO
CDK5RAP2/mypmegalin/
Cnn/Spc72/Mto1
APSB_EMENI
CNN_DROME
Q0IER0_AEDAE
F1MGF7_BOVIN
CK5P2_HUMAN
CK5P2_MOUSE
FINPH8_CHICK
E7F4R1_DANRE
MYOME_HUMAN
H2R277_PANTR
F7B2D2_CALJA
G1LZ30_AILME
F1MQ84_BOVIN
F6VAV7_HORSE
H0XE44_OTOGA
G10TX3_RABIT
MYOME_MOUSE
E
T
D
Q
I
T
Q
S
L
AER
K
L
KK
RD
I
K
65
KI
EY
T
LE
K
D
A
S
W
DL
VR S Y
QI
A AE
Q
Q
SK
L
T
R
E
E
D
DN
A
S
H
A
Q
A
70
A
T
Q
S
L
QE
ET
M
K
N
G
K
D
N
L
QES
R
D
K
I
V
R
H
G
H
N
F
R
Y
CM1
FN
E
I
A
I
L
S
60
Q
KR
R
SN
D
E
75
L
T
S
A
H
Q
S
V
Y
65
Q
K
S
K
TI
G
L
R
V
30
N
N
RSK
Y
S
NS
N
D
V
V
H
E
55
EK
A
KQ
D
H
H
Q QL T
L
KE
RSN
VK
R
H
R
R
T
A
E
K
M
Q
H
ED
M
M
D
N
V
A
D
R
B
E1BKZ0_BOVIN
V
H
T
* *
A
A
L
DS VM
SPM
R
C
D
60
*
NV
F
L
L
RR
Y QQ
REES
AK
D
E
V
T
*
KDQ
L
I
K
N
Q
M
Q
40
E
Q
G
A
E
E
Q
55
K
D VI
LE
D
Q
SIR
EQK
QS V
Y
I
D
K
N
E
Y
T
KKL
L
K
K
HR
R
R
V
I
E
I
N
S
40
KQ
L EFI
L L
L
A
T
QV
M
*
N
S
Y
NN A
C
I
V
I
30
*
VRQ
S
A
F
D
L
N
P
T
*** ** *
DQ
EG
T
NLF
H
T
K
RS
R
25
RLQVR
S
NA
T
L
D
N
R
D
T
N S
35
L
KF
LRS
Y
I
F
Y
**
E
K
K
L
R
L
N
G
QM
V
I
Q
20
A
H
E
K
G
*
Y
M T
L KQ T YKIK
E
N
KS
K
L
VD
N
Q
N
30
CK S
I
NE
LA T
VR
M
K
P
F
A
I
N
E
K
D
K
H
*
RLE
RA
I
Q
LT
EE
S
L
D
G
AG
N
Y
SQQMRRR
DD
K
A
Q
M
E
15
K
Q
Q
NDA
Y
L
F
25
W
G
KS
*
IE
D
SE
V
NRE
S
T
G
H
R
VM
L
E
*** **
S
D
K
N
RK
A
M
10
Y
M
I
20
LN
S
ET
R
S
**
A L M
V M V
I
L ENY LKIK L
Q
R
S
S
K
3.0
1.0
S
QM
E
M
E
D
AH
Y
I
N
I
N
I
N
A
I
T
I
V
K
5
4.0
2.0
Y
***
E
H
V
R
Q
V TE
GA
H
H
N
V
*
K
I
K
LQ
E
N
D
K
Q Y
D
15
*
KEQEQ
L
1.0 NSI
bits
Short CM1
(Spc72s)
2.0
G
D
R
V
10
4.0
3.0
D
E
I
N
V
5
A
Q
K
*** **
Full CM1
(Spc110s)
D
TE
E
I
A
N
S
K
ER
K
Q
NH
KV
F
L
TY
F
I L KEN LKLRKIHFLEE L
4.0
bits
Full CM1
(Pfam)
70
L
Y
KK
NNF
L
A
E
S
E
R
I
N
R
K
R
S
D
L
C
N
S
75
EN
D
N
K
S
G
H
Q
T
E
L
I
C
P
Q
S
T
Y
Figure 9 - Lin et al.
A
GCP receptor C-terminal motifs
bits
4.0
MASC
(Mto2_bdg
in Pfam)
W
3.0
2.0
RL ELE RLK ERE R
K
N
LH
QQ
E
E
A
R
Q
T
H
M
D
V
R
5
T
A
R
S
Y
N
M
A
S
IM
QK
Y
K
F
MM
TK
A
K
I
0.0
KW A
R
R
IEDM
KLIL
1.0 R
S
T
Q
Q
10
S
V
K
K
T
H
S
20
R
E
KG
V
A
D
I
Q
E
Q
A
N
S
A
S
bits
E L
3.0
MK
2.0
D
K
A
S
E
N
D
L
R
I
S
KL QE
R
K
R
E
D
TQ
A
C
SE
HKR F
K
V
E
LL
NA
Q
R
K
L
0.0
R
ITK
1.0 E
A
SL
VE
VIQ
T
A
I
Q
5
R
M
L
K
S
Y
bits
2.0
1.0
PACT
(Pfam)
0.0
G
K
R L
K
G
N
K
E
A
V
I
M
V
T
V
M
Q
AC
L
KY
LY R
W
A
YIR
KG
Q
Q
K
RK
ME
R
E
A
H
M
F
M
N
A
5
bits
3.0
L
V
S
V
R
L
T
Q
L
L
C
A
V
F
RL VRR
KM
Y
K
A KE
S
W
M
N
L
DG
R
30
K
A
I
A
20
C
K
25
M
V
S
V
EM
G
E
Q
E
N
K
30
35
**
*
K
A
Q
NEA
K
I
Y
L
V
T
R
K
L
Q
R
V
N
40
R
E
SQ
K
EH
QN
R
D
E
Y
F
V
G
Q
L RI
T
Y
T
L
L
T
F
N
M
F
K
F
50
P
S
T
A
A D
S
V
R
P
S
F
R
G
A
P
A
G
T
S
E
H
E
R
L
T
T
60
G
A
S
DL
K
S
K
Q
E
R
P
A
E
L
V
Q
K
R
H
R
R
D
I
A
F
L
G
I
S
G
L
G
P
S
E
T
R
T
S
A
P
G
N
R
S
H
R
R S K
R T
K
H
P
T
S
K
Q
R
P
S
K
N
P
P
Q
E
FRK
R
FT
P
GR
Q
V
V
S
KL
R
K
A KP
KL
P
R
D
T
V
45
D
DQ
Q
55
IA IM G
L
L
A
RM
SA
I
KE
A
I
N
T
Q
50
Q
E
A
G
T
NV
SFM
TK
NG
K
Q
D
T
L
D
D
L
T
R
RK
N
Q
CE L
N
I
D
A
S
K
D
V
K
I
Y
M
45
S
L
QS
V
T
S
H
E
D
I
A VD T
T
E
I
I
R
V
L
I
KKH
40
F
R
QMQ
K
F
VS
IN
VC
L
AS
Y QE
LIGGR
L
LI
Q
N
E
45
I QL TH VL KAR NLE
H
Q
35
R
FME
G
A
H
L
S
G
M
V FD
RD
A
H
T
N
V
V
QF
Q
Q
R
T
N
T
VF
WS
E
G
S
R
S
RQ
KLSE
V
A
S S RA
L
N
L YQ YL L L
A
R
A
Q
V T
M
A
M
R
H
Q
T
S
S
K
I
I
A
Q
T
S
A
TV
AF
C
K
W
I
R
L
I
55
60
65
70
75
80
H
Q
A
R
E
Y A
AN
L
I
Q
I
AK
S G
I
Q
I
T
25
DG
S
K
K
H
Q
T
V
AKH
S
V
S
A
RTA
KR
EL
V
FD
L A
S
E F
K
V
15
L
V
TA
I
A
M
T
F
G
T
A
M
AY
G
T
E SL K A
A
D
T
E
H
AI MRK
V VI
R
1.0 V
RAI
VS
AL
F
MS
S
A
0.0
85
L
I
W
C
C
A
E
H
I
R R
L
F
S
F
I
10
4.0
2.0
L
L
L
F
H
T
V
F
S
20
R
I
L
Q
E
S
E
K
M
EV
Q
15
4.0
3.0
N
ED
S
A
QL
R
K
10
LQ T L N Q KK MEK
KK
TKR
H
SR
A
SG
T
40
4.0
CM2
Q
N
R
A
D
35
EE
Q
K AK
V
S
QQ
KK
S
M
SV
Q
L
R
R
QD
H
R
R
K
S
K
N
V
30
L
E
RE
A
RD
R
N
GA
A
D
QR
RQ
AKF
25
EN
L
I ELE
RK
Q
EE
ES
D
D
Y
S
G
A
AN
NQ
S
NM
V
L
K
K
D A
K
Q
I
R
G
Q
LER
L
R
AE
15
A
N
W
T
R
D
M
Y
90
95
*
S
N
**
*
** **
bits
4.0
3.0
LQD
NE
2.0
1.0
PACT
(Spc110s)
Q
K
D
N
SA
SH
V
T
R
H
NDEV
L
N
Y
FR RLR
QAS
L
N
5
3.0
2.0
1.0
0.0
* * ** *
L
KL
TM
A
K
P
I
M
S
V
Q
I
Y
85
D
A
D
I
H
K
R
I
T
K
S
Y
L
V
M
T
SY
T
Y
LL
Q
I
Q
N
Q
K
TST
I
N
M
SQ
25
V V
N
MN
T
L
I
A
I
LL
V
T
V
D
K
S
30
S
KA
VLR
N
R
I
K
S
A
I
S
L
G
FL
E
I
A
E
M
Y
MND
V
FI
FR
E
SY
S
20
MT
35
R
D
LKL
I
Q
D
D
NQ
EYM
A
A
L
Q
T
40
A
G
K
T
E
N
N
I
V
Y
R
DS
TV
VNE
D
RS
N
KL
I
RDNLL
K
A
L
E
R
FM
H
N
TA
G
KK
S
N
FQ
Q
T QH
S
I
N
L
A
SG
M
K
A
G
K
V
Q
T
I
V
R
S
A
K
T
45
Y
Y
F
H
I
N
R
S
D
P
50
F
Y I D Y YP
Y
D
P
D
N
R
E
P
Y
F Y
R
T
N
R
V
55
E
D
N
L
H
K
P
Q
R
Y
V
R
S
N F
D
F
L
A
E
R
N
S
E
L
R
S S
C
A
I
M
T
G
G
N
K
P
L
Y
F
R
D
I
F
K
T
T
FA
A
A
D
L
SS
Q
N
D
Q
I
95
B
C-terminal motifs
Acentrosomal/
cytoplamic side of SPB
Acentrosomal/
cytoplamic side
of SPB
Centrosomal/
nucleus side of
SPB
Fungi and
metazoa
Ascomycota
metazoa and
Basidiomycota
CM1 only
Metazoa
Fungi and metazoa
MASC
CM2
PACT
CM1 + SPM
PotenƟal CM1 + SPM
No clear CM1 or SPM
Centrosomal/
nucleus side of SPB
Fungi
CDK5RAP2,
ApsB, Spc72,
myomegalin,
Mto1
Cnn
Spc110, Pcp1
-
Q
G
P
N
65
LR
T
E
K
SE
KY
E
NV
D
AS
Q
H
M
90
K
R R R
L
S
L
Y E
T
G
S
Q
T
KR
RA KR
HR
IR
Q
T
K
V
L
LV
R
M Y F
L
R
PCNT
AKAP9, D-PLP
P
G
A
K
Q
S
Y
V
D
60
***
SS
VK
Q
Q
H
A
M
K
15
A FVLA VRM
F
S
N-terminal motifs
bits
4.0
K
F
M
L
K
M
G
10
N LK
N
H
K
RAR
R
E
A
D
F
H
T
N
E
Q
VY
L
R
H
Q
LY
T
E
N
Y
L
ET
I
I
R
L TM
S
S
A
K SEF
Q
S
TK
M
Q
YYK KY
Q
RL
I
YN
F
M
R
D
I
T
E
SL
K
I
EY
0.0
Y
G
H
D
Q
E
K
K
R
F
E
T
P
K
FK
T
LK
G
K K
R R R PS
E
N
P
P
S
Q
70
E
G
I
N
Q
Q
SI
E
G
P
T
K
N
R
75
V
TL
G
A
I
LA
R
E
I
S
K
N
V
80
Figure 10 - Lin et al.
A
Late G1
Early S
J-TuSC oligomerization
MT nucleation
MTs
Nucleus
Duplicat plaque
Nuclear
membrane
mSPB
Cytoplasm
Bridge
mSPB
dSPB
Late S
mSPB
dSPB
M
Bipolar spindle
MT nucleation
Anaphase onset
MT nucleation inactivated
B
Tub4
Spc97
Promote new template formation
C
Tub4
Spc98
Spc97
SPM
5P
?
Spc110
Mps1
Cdk1-Clb5
Spc98
CM1
Mps1
Cdk1-Clb5
Spc110
Early S
Late S and early M