Biochemical characterization of the proteins of

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Biochemical characterization of the proteins of
Biochemical characterization of the proteins of Paramecium secretory
granules
STEPHEN H. TINDALL*, LYNN D. DEVITOf and DAVID L. NELSON*
Department of Biochemistry, College of Agricultural and Life Sciences, University of Wisconsin-Madison, 420 Henry Mall, Madison, WI53706,
USA
* Present address: Schering Corporation, Biotechnology/Biochemistry, 200 Orange Avenue, Bloomfield, NJ 07003, USA
f Present address: Department of Surgery, University of Wisconsin Medical School, Clinical Sciences Center F4/315, Madison, WI 53706,
USA
| Author for correspondence
Summary
The proteins of trichocysts (secretory granules)
from Paramecium tetraurelia have been biochemically characterized. Two-dimensional electrophoresis revealed 34 major components and at least 120
minor components, most with molecular weights
ranging from 14000 to 21000 and isoelectric points
ranging from 4-8 to 5*2. Comparison of two-dimensional electrophoretic patterns of trichocysts before
and after exocytosis revealed only minor changes in
these patterns, although the protein matrix undergoes a striking change in morphology. To clarify
the interrelationships among trichocyst proteins,
two proteins from extruded trichocyst matrix were
purified to homogeneity and sequenced at their
N termini. Their sequences are distinct, but they
share limited homology.
Introduction
granules of metazoan cells (Caulfield et al. 1980; Zuccarello, 1980; Michael et al. 1987). Second, discharge of the
trichocyst matrix into the external medium is accompanied by an eightfold increase in the body of the
trichocyst matrix length and a twofold decrease in body
diameter (Bannister, 1972). Electron microscopy of condensed and extruded trichocyst matrices reveals a striking, highly regular lattice in both (Hausmann, 1978;
Peterson et al. 19876; Sperling et al. 1987). The third
difference between trichocysts and the secretory granules
of higher organisms is that Paramecium mutant cell lines
containing altered trichocysts are available for detailed,
comparative study. Numerous mutants have been isolated that exhibit varying degrees of defective trichocyst
assembly and discharge (Pollack, 1974; Ruiz et al. 1976;
Aufderheide, 1978; Planner et al. 1980), but their
trichocysts have not been thoroughly characterized biochemically.
Trichocysts, the secretory granules found in the unicellular eukaryote Paramecium tetraurelia, were first described by Ellis (1769). Approximately 1000 membraneenclosed condensed trichocysts are attached to the inner
surface of the cell membrane in an orderly array. (We
refer to the whole organelle, with the body and tip
sheaths, and the enclosing membrane, as the trichocyst;
and to the secretory protein contents of the trichocyst as
the matrix. Trichocyst matrices are referred to as condensed matrices before exocytosis and as extruded
matrices after exocytosis.) Many other protozoans contain similar secretory organelles (Hausmann, 1978). The
carrot-shaped condensed trichocyst matrix averages
l;UmX3-7;Um and is divided into two domains, the tip
and the body (Bannister, 1972). Upon appropriate mechanical or chemical stimulation, the trichocyst contents
(matrices) are discharged into the external medium
through a Ca 2+ -mediated series of events (Hausmann,
1978). The function of these organelles has not been
defined.
Trichocysts differ in at least three ways from many
other secretory granules. First, the proteins of the
trichocyst matrix are organized in a very stable, highly
ordered array (Hausmann, 1978). Such structures have
been observed in a limited number of other secretory
Journal of Cell Science 92, 441-447 (1989)
Printed in Great Britain © The Company of Biologists Limited 1989
Key words: secretion, exocytosis, trichocyst, Paramecium.
Little is known about the proteins comprising the
trichocyst matrix. Jakus (1945) performed the initial
chemical stability studies of the trichocyst matrix. The
first biochemical characterization described matrices as
being composed of a single, acidic 17 000M r protein
(Steers et al. 1969). Subsequent studies have indicated a
greater complexity of the subunits (Adoutte et al. 1980;
Tindall, 1986). Calmodulin has been reported to be a
major component of the trichocyst (Rauh & Nelson,
441
1981). Recently, certain trichocyst matrix proteins have
been shown (Peterson et al. 19876) to be immunologically related to chromogranin A, the major protein
component of chromaffin granules from bovine adrenal
medulla cells. To determine whether the similar size and
isoelectric points of the many trichocyst matrix proteins
reflect similar (or identical) sequences, and whether the
trichocyst matrix proteins show sequence homology with
chromogranin A, we have isolated and partially
sequenced two trichocyst matrix proteins. We have made
detailed comparisons of the proteins of condensed and
extended trichocyst matrices to determine whether the
remarkable structural rearrangement of trichocyst matrix
body during secretion involves changes in the protein
subunits themselves. We have also re-investigated the
association of calmodulin with trichocysts.
Materials and methods
Trichocyst matrix isolation
Cells were cultured in axenic medium and harvested as described by Rauh & Nelson (1981). Trichocyst discharge was
induced by suspending cells in cold (4°C) Dryl's solution (Dryl,
1959) and, after low-speed centrifugation, a fluffy layer of
trichocysts and debris was present above the cell pellet. The
material in the fluffy layer was washed in lOmM-Tris-HCl,
3 mM-EDTA (pH7), then loaded onto a sucrose step gradient
(Adoutte et al. 1980) where it banded at the 55 %/58 % (w/w)
sucrose interface. Condensed trichocyst matrices were isolated
according to Peterson et al. (1987a), using the same buffer as
described above. Viewed in the electron microscope these
preparations consisted of trichocyst matrices that had lost both
the body sheath and the enclosing membrane. No other
particles or debris were visible in the light (phase-contrast) or
electron microscope.
Electrophoretic methods
The 10cm long IEF (isoelectric focusing) gels used in onedimensional analyses contained 8M-urea, 6% T acrylamide,
3 % C bisacrylamide, 3 % Bio-Lyte 4/6, and 1 % Bio-Lyte 3/10,
and were run for 18 h at 200 V and for an additional 1-5 h at 400
V (Tindall, 1986). The IEF tube gels used for two-dimensional
polyacrylamide gel electrophoresis (PAGE) contained 8M-urea,
6% T acrylamide, 12% C dihydroxyethylene-bisacrylamide,
1-2% Bio-Lyte 4/6, 0-8% Bio-Lyte 3/10, and 75 mM-Mops.
The details of the two-dimensional methods are reported
elsewhere (Tindall, 1986). SDS-PAGE for immunoblots was
performed using the method of Laemmli (1970).
Immunological methods
Male Balb/c mice were immunized with whole-cell calmodulin
that was isolated from P. tetraurelia cells as described by Rauh
& Nelson (1981). This antiserum reacted with Paramecium
calmodulin but not with bovine calmodulin, troponin C,
parvalbumin or other Paramecium proteins (DeVito, 1985).
Immunoblots
SDS-polyacrylamide gels were first soaked in 25 mM-TrisHC1, 192mM-glycine, pH8-6, for 20min and were then layered
between two sheets of 7 % acetic acid-soaked 0'45 fim nitrocellulose paper. Approximately 5-10% of the protein in the gel was
then vacuum-transferred to the membrane. The nitrocellulose
sheets were blocked with 3 % BSA (bovine serum albumin) in
442
S. H. Tindall et al.
TBS (Tris-buffered saline: lOmM-Tris-HCl, ISOmM-NaCI,
p H 8 6 ) and were subsequently incubated with serum raised
against Paramecium calmodulin. After a wash with 0-05%
Tween-20 in TBS, the nitrocellulose sheets were incubated
with alkaline phosphatase-linked goat anti-mouse IgG antibodies and were subsequently visualized using the chromogenic
alkaline phosphatase substrate 5-bromo-4-chloro-3-indolylphosphate.
Protein purification
Approximately 100 mg of sucrose density gradient-purified
discharged trichocyst matrices were incubated for 20min at
35°C in 8M-urea, 25mM-Mes, 1% (v/v) 2-mercaptoethanol,
pH 6, and were then dialysed overnight at 4°C against column
buffer (6M-urea, 25 mM-Mes, lOmM-2-mercaptoethanol,
pH6). The dialysed sample was applied to a 50ml DE-52
(Whatman) column at 4°C, which was subsequently washed
with one column volume of column buffer. The bound protein
was eluted with a 400 ml linear gradient that contained 0-0-2 MNaCl in column buffer. The column effluent was monitored at
280 nm and the conductance of the column effluent was used to
calculate the salt profile. Fractions were pooled according to
their IEF profiles and stored frozen. The weight of protein in
these fractions was quantified by the method of Bensadoun &
Weinstein (1976). All urea used for column chromatography
was treated with activated charcoal and mixed-bed exchangers,
as has been described (Tindall, 1986), to minimize carbamylation of lysines.
Preparative IEF was carried out at 13°C on milligram
quantities of DEAE-cellulose chromatography fractions. Preparative IEF samples were dialysed overnight at 4°C against
6M-urea, 10 mM-Mops, 10 mM-2-mercaptoethanol at pH 7 and
were then mixed with other gel components until the final slurry
also contained washed Sephadex G-200sf, 6M-urea (Schwartz/
Mann, Ultrapure), 1-2% Bio-Lyte 4/6 and 0-8% Bio-Lyte
3/10 (Bio-Rad), 100 mM-Mops, and 1 % 2-mercaptoethanol in a
total volume of 20 ml. The consistency of the bed was adjusted
with dry Sephadex G-200sf. The electrode buffers were the
same as for IEF in polyacrylamide gels (Tindall, 1986). Bands
were located using paper contact prints that were stained with
Coomassie Brilliant Blue G-250 in perchloric acid (Reisner et
al. 1975). Regions of gel containing bands of interest were
electroeluted in lOmM-Tris-HCl at pH8-2. Urea was removed
from these final fractions by dialysis at 4°C against 10mMNH4HCO3, p H 7 9 . The purified trichocyst matrix proteins
remained soluble under this final condition.
Protein sequencing
Gas-phase protein sequencing of trichocyst matrix proteins was
carried out by the method of Hewick et al. (1981) using an
Applied Biosystems model 470A gas-phase sequencer at the
University of Wisconsin Biotechnology Center.
Results
Changes in protein composition after exocytosis
Trichocyst secretion does not cause a dramatic alteration
in the matrix's protein composition. Two-dimensional
analyses of condensed and extruded trichocyst matrix
proteins revealed very few differences in the protein
patterns before and after exocytosis (Fig. 1). The most
noticeable difference associated with exocytosis was the
reduction in spot 30 (Fig. 1); a few other spots were also
somewhat diminished in intensity. The presence of basic
- 92 - 66 - 45 -
- 31 14
17
e 10
1011
I ;
12
5-5
PH
22. 24
M r xicr 3
32,
- 21 .^27
XL A',
,30
31
J
34
- 14 -
•
4-7
•
5-5
4-7
PH
Fig. 1. Two-dimensional PAGE of condensed (C) and extruded (E) trichocyst matrices. The arrows indicate the locations of
the major spots. Many of the 150 spots visible on the original gels are minor spots that did not reproduce well in photographs.
Each gel contains 75 fig of protein.
proteins in the trichocyst matrix was investigated using
non-equilibrium pH-gradient electrophoresis, NEPHGE
(O'Farrell et al. 1977), as the first dimension of the twodimensional analyses (data not shown). Approximately
20 additional minor spots and no additional major spots
were observed. The gels of condensed trichocyst matrices
contained five minor spots not prominent in gels of
extruded trichocyst matrices (data not shown).
Isolation of subunits
Isolation of individual subunits was accomplished by
combining DEAE-cellulose ion-exchange chromatography in urea with subsequent preparative IEF in urea.
Neither technique alone provided pure subunits, but
DEAE-cellulose chromatography yielded less complex
fractions from which pure subunits could be obtained by
preparative IEF. Fig. 2 represents a typical pH 6 DEAEcellulose chromatography profile of all urea-soluble proteins from sucrose gradient-purified extruded trichocyst
matrices. (Fractionation of trichocyst matrix proteins
using DEAE-cellulose columns run at pH 8 resulted in
the retention of all of the proteins that did not bind at
pH 6 (fractions a, b, c, Figs 3,4), but no major changes in
relative elution positions of individual proteins were
noted. Since urea decomposition is favoured by basic pH,
and since there are otherwise no outstanding advantages
of pH 8 over pH6, preparative chromatography was
carried out at pH 6.) Fig. 3 shows the isoelectric profile of
proteins present in each of the pooled regions. Approximately 60% of the material loaded onto the column was
recovered in the pooled fractions.
0-6
40
80
120 160 200
Fraction no.
240
280
320
Fig. 2. DEAE-cellulose chromatography at pH6 in 6M-urea.
The letters above the profile denote regions of the effluent
that were pooled for further purification. Fraction size was
2 ml.
The DEAE-cellulose chromatography
fractions
depicted in Figs 2 and 3 were purified further by preparative IEF in the presence of 0-lM-Mops, a pH-gradient
modifier. A representative purification of DEAE-cellulose fraction (g) gave rise to subtractions shown in Fig. 4.
This purification procedure yielded proteins that were
devoid of ampholytes, that were >95 % pure as assessed
by density scanning of SDS-PAGE and IEF gels, and
that were recovered in approximately 25 % overall yields.
From 2 to 5 fig of each of the pure proteins was obtained
permg of trichocyst matrix protein.
Paramecium secretory granule proteins
443
Basic
Acidic
as a
b
c
d
e
O
f
g
h
i
Fractions
m
n
o
Fig. 3. Composite IEF of the pH6 DEAE-cellulose chromatography fractions. The first lane contains 150 ,ug of total trichocyst
protein and the remaining lanes contain 0-5 % of the material in each pooled fraction.
N-terminal sequencing of proteins
Gas-phase sequencing of proteins 3,8, 12 and 27 (Fig. 1)
was carried out on 500 pmol quantities of intact, unmodified samples. Proteins 3 and 8 were either N-terminally
blocked or were not retained by the polybrene disk, since
< 1 0 % of the anticipated levels of PTH (phenylthiohydantoin) residues was recovered in the initial cycles.
Proteins 12 and 27 gave > 9 0 % of the expected level of
PTH residues in the first cycle and had repetitive
yields/lags of 94 %/4 % and 91 %/2 %, respectively. The
sequences of more than 25 residues were obtained for
both proteins (Table 1), and are presented in their
optimal alignment without allowing gaps. The introduc-
tion of gaps does not significantly enhance the alignment.
Since cysteine residues were not protectively modified,
some unknown residues may be cysteines. The sequences
were unrelated to any sequences within Paramecium
calmodulin (Schaefer et al. 1987), and also differed
completely from chromogranin A except for one similarity: the penultimate proline. No significant homology
with any protein in the Protein Identification Resource
data bank was detected.
Calmodulin content of gradient-purified trichocyst
matrix
Rauh & Nelson (1981) reported that calmodulin rep-
Table 1. Ammo acid sequences of proteins studied
Trichocyst protein 3*
Trichocyst protein 8*
Trichocyst protein 12*
Not detected
Not detected
SPLDTIKGVLDNFKSAVAQXQGXXDEVY
Trichocyst protein 27*
100K Chromograninf
85K Chromograninf
6SK Chromograiiinj75 K Chromograninj(chromogranin A)
Mature chromogranin A$
DPLDRLLSTLTDLEDRYVAEQKEDDAKNQ
SPVNIHMNKGEVEVMKXIVE
MPVDIDNHNEEVVTHKILEVLXNAL
MPVNIPMNKGEVEVMKIIVE
LRVNSPMNKGDTEVMKCIREVISD
LPVNSPMNKGDTEVMKCIVEVISDTLSKP
The one-letter code for amino acid sequence is used.
•Determined here.
f Reported by Settleman et al. (1985); K = 10 3 M r .
| From DNA sequence. Reported by Iacangelo et al. (1986) and Benedum et al. (1986).
444
5. H. Tindall et al.
Basic
f
1
Acidic
LLJ
<
LJU
d
e
f
g
h
Fractions
a
Fig. 4. Analytical IEF of fractions generated by preparative
IEF of DEAE fraction g. The first lane contains 20 \ig of the
original sample (fraction g) and the remaining lanes contain
1 % of the material in each pooled fraction.
resents 1-10% of the protein of crude trichocysts, and
Momayezi et ah (1986) and Garofalo (1983) provided
cytochemical evidence for the association of calmodulin
with trichocysts in situ. We used serum raised against
purified calmodulin from Paramecium to determine
whether calmodulin remained associated with thoroughly
washed trichocyst matrices after purification on sucrose
gradients. Extended trichocyst matrices isolated from
sucrose gradients and washed with buffer contained no
calmodulin detectable with this serum on immunoblots
(Fig. 5). These preparations were also devoid of the body
sheath material and membrane fragments, which are
removed during centrifugation through sucrose (J. B.
Peterson, personal communication). In positive controls,
the anti-calmodulin serum easily detected amounts of
added calmodulin representing 1 % of the protein in
trichocyst matrices (Fig. 5). Given the detection limits in
the immunoblots, immunologically reactive calmodulin
could account for no more than 0-05 % of the protein of
gradient-purified trichocyst matrices.
Discussion
Two decades ago the trichocyst matrix was thought to be
composed of a single, acidic protein given the name
2
3
4
5
Fig. 5. Immunoblot of condensed and extruded trichocyst
matrices with and without exogenously added Paramecium
calmodulin. Lanes 1 and 2 contain 100 ,ug of condensed
trichocyst matrices. Lanes 4 and 5 contain 100 ^g of extruded
trichocyst proteins. Lanes 2, 3 and 5 also contain 1 jig of
exogenously added calmodulin.
'trichynin' (Steers et al. 1969). Inspection of the twodimensional gels of the trichocyst matrices discussed here
reveals more than 150 protein spots, 34 of which can be
classified as major constituents. The bulk of these proteins have molecular weights ranging from 14000 to
21 000 and isoeleetric points in 6 M-urea ranging from 4-8
to 5-2 (Tindall, 1986), uncorrected for the effect of urea.
(The isoeleetric points of proteins determined in urea
cannot be directly compared with those determined
under native conditions. See Tindall (1986) for a description of this phenomenon.)
Purification of monomeric trichocyst matrix subunits
was complicated by the remarkable stability of the
organelle, which cannot be dissociated into its subunits
through 'mild' means such as heating, salt extraction,
treatment with non-ionic detergents, or treatment with
low levels of denaturants such as LiCl, urea or guanidine
hydrochloride (J. B. Peterson, personal communication).
The choice of urea-containing buffers permitted the
application of ion-exchange chromatography and preparative IEF as purification techniques. When combined,
these procedures yielded pure proteins that were soluble
at neutral pH and room temperature.
It had been reported that calmodulin constitutes from
1 to 10 % of the total trichocyst proteins (Rauh & Nelson,
1981). The immunological results presented here and the
calmodulin-dependent phosphodiesterase assay data (L.
D. DeVito, unpublished observations) argue that calParamecium secretory granule proteins
445
modulin is not a major component of the gradientpurified trichocyst matrix. Given the limits of detection
in these experiments, calmodulin could account for no
more than 0-05 % of trichocyst matrix protein.
Maleki et al. (1987) suggest a role for calmodulin in the
formation of exocytotic openings, and Satir et al. (1988)
have postulated a role for calmodulin at the trichocyst
membrane in controlling the access of Ca 2+ to the matrix.
Garofalo (1983) localized calmodulin in trichocysts by
immunocytochemistry at the light-microscopic level,
and by electron-microscopic immunocytochemistry,
Momayezi et al. (1986) localized calmodulin to the
periphery of condensed trichocysts in situ. They suggested that calmodulin was associated with the trichocyst
membrane, but their data would appear to be equally
consistent with calmodulin localized in the sheath inside
the membrane. Condensed trichocyst matrices in situ
contain about their periphery a net-like layer of material
(the sheath) and an enclosing membrane. The sheath is
largely lost during trichocyst matrix extension, and
appears absent from gradient-purified trichocysts (J. B.
Peterson, personal communication). A re-examination of
the original data from Rauh & Nelson (1981) reveals that
the trichocyst matrix preparations most enriched in
calmodulin were those least extensively washed, i.e. not
purified by sucrose-gradient centrifugation. It seems
probable that the difference between our present findings
and the findings of Rauh & Nelson (1981) is due to the
removal, by washing and centrifugation in sucrose gradients, of differing amounts of loosely associated material
from the sheath or membrane, the structures that exhibit
cross-reaction with antibodies against calmodulin (Garofalo, 1983; Momayezi et al. 1986). The whole condensed
trichocyst, with intact sheath and membrane, has not
been isolated; the conditions necessary to prevent extension (very low Ca 2+ concentration) apparently destroy
the membrane and remove the sheath material. It is
therefore difficult to test by isolation whether calmodulin
is part of the intact secretory granule.
The absence of calmodulin from gradient-purified
trichocyst matrices rules out that protein as a major
structural element in the trichocyst shaft, and it is
presumably not directly involved in the Ca 2+ -triggered
structural expansion that accompanies exocytosis, as that
process occurs in vitro with gradient-purified condensed
trichocyst matrices (Garofalo & Satir, 1984). One possible explanation for the dramatic structural rearrangement is some covalent modification(s) of trichocyst
protein(s). We found, however, little evidence of covalent
modification of trichocyst proteins; condensed and extended matrix materials show very little difference in
two-dimensional gel electrophoresis, which would easily
reveal charge-altering modifications such as phosphorylation. It seems likely that some non-covalent change,
such as the binding of Ca 2 + , forces the structural
rearrangement.
The interrelationships among trichocyst matrix proteins were investigated by attempting to carry out
N-terminal protein sequencing on four purified subunits.
Two of these attempts were successful. These two
trichocyst protein sequences have limited similarities
446
5. H. Tindall et al.
with each other; they are identical at six of 25 positions.
Another feature of these sequences is the occurrence of
proline at position two, which is also a common feature of
several chromogranins (Iacagelo et al. 1986; Benedum et
al. 1986). The two sequences determined here do not
otherwise share sequence homology with the known
chromogranin sequences. Although the significance of
proline at position two has not been reported, this proline
may form part of a site recognized by processing
proteases during maturation of the proteins.
Clearly, the trichocyst matrix is composed of a complex, possibly interrelated, group of proteins. The observed complexity could result from the actual presence
of many different primary sequences. Alternatively, the
complexity could result from proteolytic processing during secretory protein maturation (Adoutte et al. 1984) or
from post-translational modifications of a limited number
of primary sequences. It is not possible at this time to
distinguish how many different primary sequences are
present or whether any subunits have been post-translationally modified. As described previously (Tindall,
1986), numerous steps have been taken to ensure that
artifacts due to carbamylation by urea contaminants did
not take place. Should carbamylation occur, one would
expect to see rows of spots at given molecular weights as
described by Anderson & Hickman (1979). Furthermore,
one would expect to see an alteration of the twodimensional gel pattern as a function of sample preparation method and/or sample age. Such alterations have
not been observed.
The trichocyst system has at least one unique feature to
aid in the study of secretory granule protein structure and
function: the existence of well-characterized Paramecium
trichocyst mutants (Pollack, 1974; Ruiz et al. 1976;
Aufderheide, 1978; Planner et al. 1980). An understanding of the genetically manipulatable trichocyst system
may provide insight into the functions of similar secretory granule proteins in higher organisms.
We thank Dr Ron Niece of the University of Wisconsin
Biotechnology Center (supported by NSF grant DMB 8514305)
for determining the amino acid compositions and sequences,
the NBRF Protein Identification Resource for access to their
data bases and programs, Dr Jocelyn Colquhoun for her helpful
discussions, and Dr Joan Peterson for critical reading of the
manuscript. This work was supported by NIH grants GM
31098 and GM 32514. S.H.T. was a recipient of NIH NRSA
GM 09361.
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(Received 26 August 19SS - Accepted, in revised form,
2 December 19SS)
Paramecium secretory granule proteins
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