Nobutaka Hirokawa and Yasuko Noda

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Nobutaka Hirokawa and Yasuko Noda
Physiol Rev 88:1089-1118, 2008. doi:10.1152/physrev.00023.2007
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Physiol Rev 88: 1089 –1118, 2008;
doi:10.1152/physrev.00023.2007.
Intracellular Transport and Kinesin Superfamily Proteins, KIFs:
Structure, Function, and Dynamics
NOBUTAKA HIROKAWA AND YASUKO NODA
Department of Cell Biology and Anatomy, Graduate School of Medicine, University of Tokyo, Tokyo, Japan
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I. Introduction
II. Classification
III. Anterograde Axonal Transport
A. KIF1A/Unc-104: a monomeric motor for synaptic vesicle precursor transport
B. KIF1B␤: a second monomeric motor for transport of synaptic vesicle precursors
C. KIF1B␣: a monomeric motor for mitochondrial transport
D. KIF5: a major dimeric motor for axonal transport
E. KIF3/Kinesin-II: a heterodimeric motor for axonal transport
F. KIF13B/GAKIN
IV. Dendritic Transport in Neurons
A. KIF17: an NMDA receptor transporter
B. KIF5: an AMPA receptor transporter
C. KIF5: an mRNA transporter
D. KIFC2
E. CHO1/MKLP1
F. KIF21B
V. Conventional Transport, Including Endoplasmic Reticulum to Golgi, Lysosomes, and Endosomes
A. Transport between the endoplasmic reticulum and Golgi apparatus
B. Lysosomal transport
C. Transport from the Trans-Golgi network to the plasma membrane
D. Endosomal recycling
VI. Slow Axonal Transport
VII. Polarized Sorting by Motor Proteins
VIII. Development and Molecular Motors
A. KIF3: left-right determination and development
B. Transport of N-cadherin in developing neurons
C. KIF2: a suppressor of collateral branch formation
D. KIF4: a regulator of neuronal survival
IX. Regulation of Cargo Binding
A. Use of an adaptor/scaffolding protein complex for cargo binding
B. Autoinhibition/phosphorylation
C. Tug-of-war between motor proteins
X. Structure of Motor Proteins
A. Structure of KIFs and microtubules
B. Mechanism that couples ATP hydrolysis and conformational change
C. Processive movement of KIF5
D. Structure of C-kinesins
E. Monomeric motor KIF1A: how does it move?
F. Structure of KIF2C/MCAK: a common mechanism for microtubule destabilization
XI. Conclusions and Future Perspectives
Hirokawa N, Noda Y. Intracellular Transport and Kinesin Superfamily Proteins, KIFs: Structure, Function, and
Dynamics. Physiol Rev 88: 1089 –1118, 2008; doi:10.1152/physrev.00023.2007.—Various molecular cell biology and
molecular genetic approaches have indicated significant roles for kinesin superfamily proteins (KIFs) in intracellular
transport and have shown that they are critical for cellular morphogenesis, functioning, and survival. KIFs not only
transport various membrane organelles, protein complexes, and mRNAs for the maintenance of basic cellular
activity, but also play significant roles for various mechanisms fundamental for life, such as brain wiring, higher brain
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0031-9333/08 $18.00 Copyright © 2008 the American Physiological Society
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functions such as memory and learning and activity-dependent neuronal survival during brain development, and for
the determination of important developmental processes such as left-right asymmetry formation and suppression of
tumorigenesis. Accumulating data have revealed a molecular mechanism of cargo recognition involving scaffolding
or adaptor protein complexes. Intramolecular folding and phosphorylation also regulate the binding activity of motor
proteins. New techniques using molecular biophysics, cryoelectron microscopy, and X-ray crystallography have
detected structural changes in motor proteins, synchronized with ATP hydrolysis cycles, leading to the development
of independent models of monomer and dimer motors for processive movement along microtubules.
I. INTRODUCTION
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Cells have developed a differentiated delivery system
to sustain their specific functions and morphology. This
intracellular transport mechanism is spatially and temporally controlled by microtubule-dependent motor proteins. As shown by recent data, the basic principles of
intracellular transport are highly conserved, and motor
proteins constitute a common molecular machinery for
intracellular transport in neurons as well as in other types
of cells (65, 71, 96, 101, 229). Compared with other cell
types, in which only a short distance of transport is required to reach the destination, neurons with long neurites have a well-developed transport system. Indeed, the
transport of membrane organelles in axons can be directly observed by high-resolution optical microscopy,
which reveals that different-shaped membrane organelles
are transported at different speeds with different directionalities. Thus axonal transport and dendritic transport
serve as a good model system for elucidating one of the
fundamental mechanisms of sustaining life in organisms.
The directionality of transport is determined by interactions between motor proteins and the microtubule
rails, tubular structures 25 nm in diameter composed of
heterodimers of ␣- and ␤-tubulins (64, 65). A microtubule
has its own direction with plus and minus ends: microtubules polymerize faster at the plus end than at the minus
end, which is less dynamic. Each motor protein senses the
direction of the microtubules and steers with its own
directionality toward the determined end. Thus, to understand each type of transport, knowledge about the directionality of microtubules within cells is necessary. In
axons, microtubules are unipolar, and the plus ends always point to the periphery. Therefore, anterograde motors, which drive transport from the cell body to the cell
periphery in axons, are necessarily plus-end-directed,
whereas retrograde motors, which drive transport from
the periphery to the cell body, are minus-end-directed.
However, the directionality of microtubules is mixed in
proximal dendrites, in which both types of motor can
work (6, 18).
The polarity of microtubules also depends on the cell
type. For example, in epithelial cells, the minus ends of
microtubules are directed towards the apical surface. In
fibroblasts, microtubules radiate in various directions
from the microtubule-organizing center near the nucleus,
and their plus ends are directed towards the periphery.
Each type of cell has its own pattern of highly organized
microtubule rails and uses compatible motors along them.
In the mid 1980s, two representative motors, conventional kinesin and cytoplasmic dynein, were purified from
the brain and found to utilize ATPase activity to drive
microtubule plus-end- and minus-end-directed transport,
respectively (15, 172, 230). Initially, it was thought that
these two motors could accomplish most of the bidirectional transport in cells. In contrast, since then, a range of
cargoes steered by microtubule motors have been identified and characterized (65).
Quick-freeze, deep-etch electron microscopy of axons revealed fine structures associated with membrane
organelles and microtubules at a very high resolution.
Short crossbridges can be detected between membrane
organelles and microtubules, and these are supposed to
correspond to the molecular motors (Fig. 1) (64 – 66, 68).
Indeed, these crossbridges have different shapes, reflecting the variable shapes of molecular motors (Fig. 1, A–C).
In 1992, the first molecular biological search of a mouse
brain cDNA library identified a group of 10 molecular
motor genes, which were then designated as kinesin superfamily proteins (KIFs) (1).
Over the course of evolution, various combinations
of dynein subunits have come to constitute a large “dynactin complex” for the purpose of binding a variety of
cargoes (191, 231), while the number of kinesin family
members has increased to provide variation. Either way,
each molecular motor has attained the specificity to bind
to its partner on the cargo complex, enabling the proper
execution of a transport process based on a strict regulation mechanism.
In this review, we focus on the intracellular transport
mediated by KIF motor proteins to understand the molecular mechanism underlying the regulation of differentiated transport. Thus we do not refer to mitotic KIF
motors in this review; they have been discussed comprehensively in other reviews (133, 141, 198).
This review is composed of three parts. First, each
type of molecular transport mediated by KIF motors is
described in terms of the route taken, the particular cargoes being transported, and the particular motor proteins
involved (see sects. III-VII). At a higher level, such as in
tissues and individual animals, motor proteins have been
KIFS AND INTRACELLULAR TRANSPORT
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implicated in determining cell morphology, cell-cell contacts, and differentiation (see sect. VIII).
Second, the interactions among molecules are discussed. To execute multistage transport processes, intermolecular interactions between motor proteins and
cargoes are indispensable (see sect. IX). Third, the intramolecular mechanism underlying the conformational change in motor proteins is also discussed,
through an analysis of the crystal structures and the
cryo-electron microscopic (EMs) images of motor proteins (see sect. X).
II. CLASSIFICATION
KIFs possess a conserved globular motor domain,
which involves an ATP-binding sequence and a microtubule-binding sequence (Fig. 2) (65, 68, 71). This globular
motor domain, called the “head,” hydrolyzes ATP and
transfers chemical energy to result in the motility of each
KIF along microtubules with intrinsic directionality.
While motor domains show high amino acid sequence
homologies of ⬃30 – 60% among various KIFs, other rePhysiol Rev • VOL
gions, including a filamentous “stalk” region and a globular “tail” region, are quite variable (65, 68). Generally,
motor proteins use their stalk regions to dimerize with
each other. However, some KIFs have a short coiled-coil
region and exist as monomers, or some form heterodimers among the subfamily members. KIFs bind to
cargoes through their variable tail regions (65, 68). Some
accompany light chains or associated proteins to bind
indirectly to cargoes (Fig. 3). In addition to transporting
cargoes, motor proteins bind to chromosomes and spindles and are functional during mitosis and meiosis. Some
motor proteins participate in both intracellular transport
and mitosis.
KIFs can be broadly grouped into three types depending on the position of the motor domain within a
molecule. N-kinesins have a motor domain in the NH2terminal region, M-kinesins have one in the middle, and
C-kinesins have theirs in the COOH-terminal region (Fig.
2). The intramolecular position of the motor domain
grossly determines the directionality of the motor. While
N-kinesins drive plus-end-directed motility, C-kinesins
minus-end-directed motility.
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FIG. 1. Electron micrographs of membrane
organelles transported along microtubules in an
axon, obtained by quick-freeze, deep-etching
techniques. A–C: short crossbridges, which are
supposed to correspond to different molecular
motors (arrows), can be noted between membrane organelles and microtubules. Bar, 50 nm.
[From Hirokawa (66).]
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Recently, all KIF genes in the mammalian and human
genomes have been systematically identified (137). There
are a total of 45 KIF genes in the mouse genome, 38 of
which are expressed in the brain. Considering that alternative splicing can produce two to three mRNAs from
each gene, with different tail domains that bind to differ-
III. ANTEROGRADE AXONAL TRANSPORT
A. KIF1A/Unc-104: A Monomeric Motor for
Synaptic Vesicle Precursor Transport
Among the KIFs (Fig. 2), some members; KIF1A,
KIF1B␣, and KIF1B␤, have a short coiled-coil domain in
the stalk region, which are supposed to exist as mono-
FIG. 3. Electron micrographs of KIF
complexes observed by low-angle rotary
shadowing. KIFs are observed as single or
double globular molecules corresponding to
the complex composition. The stalk region is
sometimes visualized as filamentous structures. The right column shows molecular
models of KIFs; some are monomers, and
some form heterodimers or homodimers
with or without light chains or associated
proteins. Bar, 100 nm. [From Hirokawa (65).]
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FIG. 2. Kinesin superfamily proteins (KIFs) in intracellular transport. Conserved catalytic motor domains that involve an ATP-binding
sequence and a microtubule-binding sequence are indicated in purple.
Most KIFs have these domains in the NH2-terminal region, while others
have them in the middle or COOH-terminal regions. Members of the
kinesin-3 family specifically have FHA domains in the NH2-terminal stalk
regions (yellow). Some have PH/PX domains in the tail domain (blue).
ent cargoes, the number of KIF proteins is approximately
twice the number of KIF genes, perhaps even larger. Each
KIF protein has been identified independently and named
by different criteria, so there have been reported hundreds of KIFs containing the same KIFs called with different names, which has caused confusion and miscommunication among researchers. To improve the situation,
a standard kinesin nomenclature was established in 2004,
which classified them into 14 families according to the
results of phylogenic analyses (24, 68, 118, 119, 135, 136).
Of the 14 families, only one contains M-kinesins, and only
one contains C-kinesins; the remaining 12 families comprise N-kinesins, a bias that can be explained by the fact
that KIFs usually drive anterograde transport while most
retrograde transport is mediated by cytoplasmic dynein.
In spite of the new nomenclature, to date, its usage has
only been limited to introduce rough categories of the
motors. Indeed, each KIF is still called by its original
name, since different KIFs usually transport different cargoes, even if they are categorized in the same group. The
KIFs implicated in transporting identified cargoes are
shown in Figure 2, according to the new classification.
FIG.
Recent work has shown that KIF1A binds to its cargo
through its pleckstrin homology (PH)-domain in the tail
region, which interacts specifically with phosphatidylinositol 4,5-bisphosphate (PIP2) (109) (Fig. 5). When KIF1A
molecules formed a cluster at the PIP2-abundant membrane surface, the efficiency of the motor activity of
KIF1A increased (223). The authors of this report also
showed that recombinant chimeric KIF1A, which is designed to dimerize, transports vesicles as efficiently as
highly concentrated monomeric KIF1A. In another report,
recombinant KIF1A of higher concentration was detected
to exist as a dimer even in the soluble condition. They
insisted that presence of the short coiled-coil region is
sufficient to dimerize KIF1A (201). However, these data
do not directly indicate that native monomeric KIF1A
molecules dimerize when clustered on the membrane.
Moreover, a newly identified binding partner, liprin-␣,
colocalized with KIF1A by binding to the coiled-coil region of KIF1A (202). The degree of contribution of
dimeric KIF1A to molecular transport will need to be
determined in the future.
Functional and biological damage caused by disruption of kif1A has been reported in mouse (251). Mutant
mice were born alive, but all died within 24 h because
they did not suckle milk. Severe motor and sensory abnormalities were explained by a decrease in the number
of synaptic terminals per unit area, and in the density of
synaptic vesicles at synaptic terminals to 50 – 60% of that
seen in wild-type mice (Fig. 7). Focal neuronal death was
also observed in several brain areas. The premature death
of primary cultured hippocampal neurons within 13 days
4. Intracellular transport by molecular motors in neuronal cells.
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mers. Among them, KIF1A is a brain-specific microtubule
motor, with 1,695 amino acid residues (Fig. 2) (1, 168). It
was first identified as Unc-104 in Caenorhabditis elegans,
the mutation of which causes a deficiency of synaptic
vesicles in axons (53). A unique property of KIF1A is its
existence as a monomer, which is strongly suggested by
various experimental data (168). Sucrose velocity gradient, native polyacrylamide gel electrophoresis (PAGE),
and differential laser light scattering analyses have shown
that the native molecular mass of KIF1A in solution is 200
kDa, which closely agrees with the molecular mass of 190
kDa estimated from its amino acid sequence. Low-angle
rotary-shadowing electron microscopy also revealed that
KIF1A is a single globular molecule (Fig. 3).
A motility assay using microtubules demonstrated
that KIF1A moves towards the plus ends of microtubules
at a velocity of 1.2 ␮m/s (168), which suggests that KIF1A
is one of the fastest anterograde motors. Nerve ligation
experiments showed the accumulation of KIF1A in the
proximal region of a ligated site where anterogradely
transported membrane organelles accumulated. Further
experiments using immunocytochemistry and immunoprecipitation revealed that KIF1A-containing vesicles correspond to a particular population of synaptic vesicle
precursors that contain synaptophysin, synaptotagmin,
and Rab-3A, but do not contain plasma membrane proteins, such as syntaxin1A or SNAP25 (168) (Fig. 4). Evidence that conventional kinesin (KIF5, see sect. IIID) and
KIF3 (see sect. IIIE) are not present in KIF1A-containing
vesicles suggests independent transport of these three
motor proteins in axons.
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coincides well with the beginning of the synthesis of
KIF1A in wild-type neurons (251).
Although KIF1A is vital for neuronal function and
survival, its functions are partially compensated for in
knockout mice; about half of the synaptic vesicles density
is still observed in knockout mice. This observation suggests the existence of complementary motor proteins,
which is often the case with the intracellular transport
system.
B. KIF1B␤: A Second Monomeric Motor for
Transport of Synaptic Vesicle Precursors
KIF1B␤ is an isoform generated by alternative splicing of the gene encoding KIF1B␣. The two motor proteins
have identical motor domains, but completely different
COOH-terminal tail regions (Fig. 2), indicating their involvement in the transport of different cargoes, because
motors bind to cargoes through their tail regions. Biochemical analyses such as glutathione S-transferase
(GST)-pulldown assays and vesicle immunoprecipitation
using an anti-KIF1B␤-specific antibody have revealed that
KIF1B␤ transports synaptic vesicle precursors containing
synaptotagmin, synaptophysin, and SV2 (254), suggesting
a partially overlapping function of KIF1B␤ with that of
KIF1A (Fig. 4). When the function of KIF1B␤ was invesPhysiol Rev • VOL
C. KIF1B␣: A Monomeric Motor for
Mitochondrial Transport
KIF1B, another member of the kinesin-3 family, was
identified in 1994 and is now called KIF1B␣ (Fig. 2) (153).
Experimental data suggest that KIF1B␣ also exists as a
monomer, and by low-angle rotary-shadowing electron
microscopy, KIF1B␣ is observed as a single-head globular
molecule, sometimes with a short tail (Fig. 3). Immunocytochemistry and subcellular fractionation data showed
colocalization of KIF1B␣ and mitochondrial markers. An
in vitro reconstruction assay succeeded in visualizing the
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FIG. 5. Gallery of the molecular cargoes transported by KIFs.
Molecular complexes including adaptor proteins have been identified to
bind directly to KIFs for the transport of functional molecules.
tigated in kif1B knockout mice, in which neither KIF1B␣
nor KIF1B␤ was expressed, mice were born alive but died
within 30 min due to apnea. No significant defects were
seen in the structures of alveoli, respiratory muscle, or
neuromuscular junctions involved in respiration, suggesting the possibility that the apnea originated from neurological damage. In the brains of kif1B knockout mice, the
number of neuronal cell bodies was ⬍25% of that in the
controls. Neuronal loss in the respiratory center was so
severe that it was likely to cause neonatal apnea. The
density of synaptic vesicles also decreased to 50 – 60% of
that in controls.
When hippocampal neurons obtained from kif1B
knockout embryos were cultured, a significant proportion
of mutant cells died within 1 wk of plating (254), similar
to kif1A null cells. Because exogenous expression of
KIF1B␤ but not that of KIF1B␣ rescued the neuronal
death, KIF1B␤ is suggested to be mainly responsible for
the mutant phenotype. Unlike KIF1B␤, the absence of
KIF1B␣ in kif1B knockout mice seems to be compensated
by other motors.
Although kif1B knockout mice died shortly after
birth, heterozygotes survived. After 1 year, however, they
come to show progressive muscle weakness and motor
uncoordination (254) (Fig. 7). In the peripheral axons of
heterozygous mice, a specific decrease in the levels of
KIF1B␤ and synaptic vesicle proteins, such as synaptotagmin and SV2, was observed.
After gene mapping of murine kif1B␤ on chromosome 4E, the Charcot-Marie-Tooth disease type 2A
(CMT2A) was mapped to the overlapping human chromosome region, 1p35–36. Analysis of a pedigree of CMT2A
revealed a Q-to-L (glutamine-to-leucine) mutation in the
consensus ATP-binding site of the KIF1B motor domain in
heterozygote patients; this mutation causes a significant
decrease in microtubule-dependent ATPase activity in
vitro (254). Exogenous expression of the Q-to-L mutant
KIF1B␤ in fibroblasts also caused perinuclear aggregation
of the motor proteins instead of their transport to the
peripheral plus ends of microtubules, suggesting kif1B␤
as a causative gene for Charcot-Marie-Tooth disease type 2A.
D. KIF5: A Major Dimeric Motor for
Axonal Transport
KIF5, a conventional kinesin, was the first identified
and is the most abundant motor protein (68, 230); it has
now been revealed to consist of three closely related
subtypes: KIF5A, KIF5B, and KIF5C (Fig. 2) (1, 100).
While KIF5B is expressed ubiquitously, KIF5A and KIF5C
are neuron specific. In contrast to KIF1A and KIF1B, KIF5
proteins form homo- or heterodimers among themselves
through the coiled-coil region in their stalk domains (100).
Within cells, about half of the KIF5 proteins present are
supposed to form tetramers by recruiting two light chain
molecules (KLCs) (49). KIF5 binds KLCs through light
chain-binding domains in the stalk and tail domains (30,
49, 68) (Fig. 3). KIF5 molecules bind KLCs through the
NH2-terminal regions of these light-chain molecules; the
KLCs then bind to cargoes through their COOH-terminal
domains, which assume various forms as a result of alternative splicing (131, 176, 210). KIF5 proteins also have
specific cargo-binding regions in their tail domains which
are localized in the COOH terminus of the light chainbinding domains, (192, 206) and have the ability to bind
directly to cargoes, suggesting the existence of two forms
of transport mediated by KIF5 proteins, direct or indirect
via KLCs.
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KIF5 proteins play essential roles in axonal transport.
In Drosophila, there is only one kif5 and one kinesin
light chain gene. Therefore, Drosophila mutants of kif5
show a severe motor neuronal disease phenotype and
lethality (84, 189). Within axons, “organelle jams” stacked
with membrane vesicles and organelles were observed,
and these obstructions disrupted both anterograde and
retrograde transport. The same phenotype is also caused
by the loss of KLC in Drosophila (43). On the other hand,
in mouse, there exist three KIF5 proteins and at least two
KLCs with many spliced variants (100, 131, 176). In spite
of existence of multiple KIF5 proteins, disruption of neuron-specific KLC1 results in an aberrant pool of KIF5A in
the peripheral portion of the Golgi apparatus (177), suggesting differentiated functions among KIF5/KLC subtypes. When kif5A was conditionally targeted by a synapsin-promoted Cre-recombinase transgene, young mutant
mice showed no sign of interrupted transport within axons, but an accumulation of neurofilament in the cell
body, suggesting a role for KIF5A as a neurofilament
motor (243) (Fig. 4). This could explain the effect of a
missense mutation in kif5A in patients with hereditary
spastic paraplegia (179). In fact, this mutation disrupts
microtubule-activated kinesin activity in vitro and may
inhibit the axonal transport of neurofilament in vivo. In
contrast to KIF5A, kif5C knockout mice survive with no
abnormality except for a reduction in brain size (100).
Reflecting the relative abundance of KIF5C in motor neurons, the number of motor neurons was decreased by 28%.
As upregulation of KIF5A or KIF5B was not detected in
the kif5C knockout brain, the restricted distribution of
KIF5C may minimize an overt effect caused by depletion
of this molecule.
To investigate the intracellular functions of KIF5 proteins, depletion of these proteins from cell culture systems was performed using antisense oligonucleotides
(36). In hippocampal neurons, induction of antisense oligonucleotides against kif5 reduced the overall length of
neurites and inhibited the transport of GAP-43 and synapsin to the tips of neurites, showing specific transport of
these two molecules by KIF5 proteins. By conventional
approaches that disrupt the function of one motor and
allow the localization of known transported molecules to
be examined, we were able to determine whether each
motor is essential for transport of a specific molecule
being transported; however, since this approach requires
the knowledge of the molecule being transported, we still
have no way to find a motor for unknown molecule. New
technological progress now makes it possible to identify
new binding partners, by the aid of a mass spectrometric
approach combined with immunoprecipitation, or with
GST pulldown experiments; alternatively, they can be
identified directly by using yeast two-hybrid systems.
With the use of these methods, an increasing number
of molecules have been reported to be involved in the
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cotransport of KIF1B␣ and purified mitochondria along
microtubules at a velocity of 0.5 ␮m/s, suggesting that
KIF1B␣ is the motor involved in the transport of mitochondria (153) (Fig. 4). As hinted at in the previous section, KIF1B␣ is not the only motor involved in the transport of mitochondria. Knockout of KIF5B or KIF5C, two
closely related subtypes of conventional kinesin (100,
215), showed that KIF5 molecules are also motors for
mitochondrial transport, explaining the compensation of
mitochondrial transport in kif1B knockout mice.
Recent works have shown that the localization of
KIF1B␣ in mitochondria is controlled by the newly identified KIF1 binding protein (KBP) by yeast two hybrid
screens (241). Overexpression of the dominant-negative
protein or an antisense construct decreases the activity of
KIF1B␣ and leads to the aggregation of mitochondria in
vivo.
Other authors have reported that seven amino acids
in the COOH-terminal region of KIF1B␣ selectively interact with the PDZ domains of PSD-95, PSD-97, and SSCAM, members of a family of synaptic vesicle-associated
scaffolding proteins (139). Although their immunocytochemistry data showing a diffuse overlapping distribution
of KIF1B␣ and these scaffolding proteins in cultured neurons were not sufficiently persuasive, the decrease in the
level of KIF1B␣ could, to some degree, explain the peripheral neuropathy caused by kif1B mutation.
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Cargo molecules that bind directly to KLC have also
been identified. These usually bind KIF5 tetramers via the
tetratricopeptide repeat (TPR) domain of KLC (30). Accumulating data have revealed a particular population of
cargoes that KIF5s bind indirectly via KLCs. Among them,
c-jun NH2-terminal kinase (JNK)-interacting proteins
(JIPs) are well characterized (14, 234). In mammals, three
subtypes of JIPs (JIP1–3) have been identified (89). The
first evidence of a relation between JIPs and KIF5 proteins came from studies of Drosophila. Mutants of the
Drosophila homolog of JIP3, Sunday driver (SYD), caused
aberrant accumulation of axonal cargoes, closely resembling the phenotype of kinesin mutants. With the use of
the yeast two-hybrid method, SYD was found to bind to
the TPR domain of KLC (14). JIPs were also identified by
yeast two-hybrid analysis and immunoprecipitation during search for a binding partner of KLC (234). JIPs function primarily as scaffolding proteins to mediate the JNK
signaling cascade by directly binding to MAPK, MAPKK,
and MAPKKK (102). On the other hand, they bind to KLC
and connect ApoER2 Reelin receptor-containing vesicles
to the motor protein complex (234) (Figs. 4 and 5). The
clarification of the relation between two functions of JIPs
needs further experiments.
An antibody against amyloid-␤ precursor protein
(APP) immunoprecipitates KIF5B and KLCs. Direct binding of APP to the TPR domain of KLC has also been
recognized (95, 97) (Figs. 4 and 5). Although the specificity of binding was confirmed in KLC-1 knockout mice, no
direct interaction was detected between APP and KLC in
other experimental systems (120). As no change in the
transport driven by KIF5 was observed in APP knockout
mice, the function of APP as an adapting protein for KIF5
is now controversial. A subsequent finding that linked the
two conflicting lines of evidence showed that JIP1 enhances a weak association between APP and KLC by
binding simultaneously to APP and KIF5 (146). JIP1 is
also reported to accelerate the phosphorylation of APP by
JNK, and to assist the transport of phosphorylated forms
of APP only (88). These data may explain the limited
effect of KIF5 on APP transport.
E. KIF3/Kinesin-II: A Heterodimeric Motor for
Axonal Transport
KIF3 proteins are categorized in the kinesin-2 family.
They are ubiquitously expressed in tissues and are especially abundant in the brain. They usually exist as a heterotrimeric complex of KIF3A, KIF3B, and kinesin superfamily-associated protein 3 (KAP3) with a stoichiometry
of 1:1:1 (1, 62, 67, 111, 237, 246, 247). As KAP3 binds to the
tail domains of KIF3s (Fig. 3), KIF3s bind to cargoes
through the Armadillo repeats of KAP3. KIF3s steer not
only the transport of vesicles, but also intraflagellar rafts
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transport of cargoes by KIF5 proteins. While synaptic
vesicle precursors are conveyed by monomeric motors,
such as KIF1A and KIF1B␤, SNARE proteins, which are
essential for the docking of synaptic vesicles at presynaptic membranes, are reported to be carried by KIF5s.
Direct binding between SNAP25 and the cargo-binding
domain of KIF5 proteins was recognized using a yeast
two-hybrid system and confirmed by an in vitro binding
assay (29) (Fig. 4). Syntaxin was also found to bind to
KIF5 proteins via syntabulin, a new partner that shares
homology with the p150 subunit of the dynactin complex
(212). Syntabulin and syntaxin transport are suggested to
be independent of KLC, since syntabulin binds directly to
the COOH-terminal region of KIF5 in an in vitro assay
(Fig. 5).
As stated in the previous section, mitochondria are
also transported by KIF5 proteins. Mitochondria accumulate in the centers of cells when the kif5B gene is disrupted (215). In mice, a kif5B null mutation is embryonic
lethal, but the mitochondrial phenotype in yolk sac-derived cultured cells from kif5B null mice could be rescued
by exogenous expression of either KIF5A, KIF5B, or
KIF5C, suggesting that any type of KIF5 can transport
mitochondria separately (100) (Fig. 4). Some investigators have reported that hyperphosphorylation of KLC inhibits the function of KIF5 proteins and causes clustering
of mitochondria in the perinuclear region (25); however,
others have shown KLC-independent axonal transport of
mitochondria mediated only by milton and KIF5 (44) (Fig.
5). These authors showed a competitive interaction between milton and KLC for KIF5 using cotransfection experiments (44). KLC may function to regulate the motor
activity of KIF5, but not be essential for binding mitochondria. Recently, syntabulin was also reported to possess another binding site for mitochondria, but its relationship to milton remains to be determined (19, 44).
Using a combination of pulldown assays and mass
spectrometric analysis, the direct binding of DISC1 and
KIF5 was recently identified; these molecules then form a
cargo complex with NUDEL, LIS1, and 14 –3-3␧ (217).
Although DISC1 has been reported to function with cytoplasmic dynein rather than KIF5, the transport of this
complex by KIF5 contributes to axonal elongation in neurons.
Although an increasing number of cargo molecules
have been reported, not all components or functions have
been revealed yet. In some cases, only binding has been
recognized at present. For example, ␤-dystrobrevin, a
dystrophin-related protein, has been recognized to bind
directly to KIF5A and KIF5B (127). Other disease-related
proteins have also been reported to be associated with
KIF5 complex. While neurofibromin binds directly to
KIF5, huntingtin-associated protein-1 and torsinA bind
indirectly via KLC. Their precise function in vivo is under
investigation (52, 98, 132).
F. KIF13B/GAKIN
KIF13B, another member of the kinesin-3 family, was
first identified by an improved PCR strategy (148). Subsequently, it was also analyzed as a guanylate kinase-associated kinesin (GAKIN) by GST pulldown using the guanylate kinase-like (GUK) domain of the discs large tumor
suppressor protein (Dlg) (54). In Drosophila neuroblasts,
the transport of Dlg by KIF13B induces astral spindles at
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the apical cortex through the interaction of Dlg with Pins,
Par and Insc, which is essential for determining cortical
polarity in a microtubule-dependent manner (205).
KIF13B was also found to be a binding partner of
centaurin-␣1 by yeast two-hybrid experiments (232). Centaurin-␣1 is known as a molecule with a double function:
as a GTPase-activating protein (GAP) for the ADP-ribosylation factor (ARF), and as a phosphatidylinositol 3,4,5trisphosphate (PIP3)-binding protein. Interestingly, centaurin-␣1 binds specifically to a forkhead-associated
(FHA) domain in KIF13B, commonly locating in the NH2terminal stalk domain of the kinesin-3 motor (Fig. 2).
KIF13B recruits centaurin-␣1 to the plasma membrane of
the leading edge and regulates the activity of ARF6
through its GAP function in nonneuronal cells. In neurons, KIF13B is reported to bind PIP3 via centaurin-␣1 and
transports PIP3-containing vesicles to the tips of axons
(Figs. 4 and 5). This process seems to initiate axonal
differentiation, as overexpression of KIF13B induces the
formation of multiple axonlike neurites that contain abundant PIP3 (79). The role of KIF13B in ARF6 activation in
neurons remains to be determined.
IV. DENDRITIC TRANSPORT IN NEURONS
In contrast to axons in which microtubules run unidirectionally, with their plus ends directed to the distal
ends, the polarity of microtubules in proximal dendrites is
mixed, while in the distal dendrites, the polarity is the
same as in axons. Some dendritic motors, such as KIF17,
sense the difference between the two types of neurite and
exist predominantly in dendrites, while other motors, like
KIF5 proteins, transport cargoes in both dendrites and
axons.
A. KIF17: An NMDA Receptor Transporter
KIF17, an NH2-terminal motor domain-type motor, is
a member of the kinesin-2 family, along with the KIF3
proteins (148, 195). In contrast to KIF3 proteins, KIF17 is
mainly localized in the cell bodies and dendrites of neurons. By yeast two-hybrid assay, Lin-10 (mouse homolog
of Caenorhabditis elegans LIN-10), also known as Mint1,
was identified as a binding partner of KIF17. mLin-10 has
two PDZ domains, and the KIF17 tail domain binds to the
first of these. This interaction was also confirmed by
immunoprecipitation using an anti-mLin-10 antibody and
a BIAcore system. Via mLin-10, KIF17 was found to bind
successively to mLin-2, mLin-7, and finally to the NR2B
subunit of N-methyl-D-aspartate (NMDA)-type glutamate
receptors, all of which are members of a previously identified complex on NMDA receptor-binding vesicles (93)
(Figs. 4 and 5).
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in cilia and flagella (22). The developmental effect of
disruption to KIF3s on ciliogenesis will be discussed in
section VIIIA.
In neurons, fodrin has been recognized as a binding
partner of KAP3 by yeast two-hybrid experiments. The
transport of vesicles containing fodrin by KIF3 proteins is
essential for neurite elongation, which is blocked by microinjection of antibodies against KIF3 into cultured superior cervical ganglion neurons (213) (Figs. 4 and 5).
KIF3 proteins are also essential for the polarization
of neurons. The tumor suppressor gene adenomatous polyposis coli (APC) binds to KAP3 via an interaction that is
mediated by the Armadillo repeats of both molecules
(200). Exogenous expression of a partial deletion mutant
of KAP3 that binds APC but not KIF3 selectively inhibited
the transport of APC to the tips of protrusions, suggesting
transport of APC by KIF3 via KAP3. In neurons, APC
forms a complex with Par3, and its accumulation at the
tip of the axon is essential for the polarization of neurons.
A direct interaction between Par3 and the tail domain of
KIF3A has also been reported (154) (Fig. 5). Considering
that APC also binds to glycogen synthase kinase-3␤ (GSK3␤), a determinant of the fate of axons that is downstream
of phosphatidylinositide 3-kinase, it can be concluded
that transport of the APC complex by KIF3 proteins plays
a key role in the axonogenesis of neurons.
Furthermore, KIF3 proteins have been shown to
transport choline acetyltransferase and acetylcholinesterase in Drosophila axons. Although siRNA-mediated
knockout of each component of the motor complex disrupted the transport of both enzymes, these two enzymes
when they were labeled fluorescently were found to be
transported independently with different properties
within the same axon. Different molecular weight splice
variants of KIF3A appear to be involved in the transport of
these two enzymes (10). A recent paper showed the involvement of KIF3 proteins in the transport of axonal
voltage-dependent potassium (Kv) channels (46). Fluorescence resonance energy transfer (FRET) analysis also
revealed the cotransport of Kv␤2 and EB1 along the axon.
Whether or not the targeting mechanism of these microtubule plus-end-tracking proteins (⫹TIPS) to the plus end
of microtubules contributes to transport of these channels will be revealed by further experiments.
1097
1098
B. KIF5: An AMPA Receptor Transporter
As noted in the previous section, KIF5 proteins play
a major role in fast axonal transport. Moreover, a role in
dendritic transport has also been identified for KIF5 proteins. Yeast two-hybrid assays using the cargo-binding
domain of KIF5 as bait identified glutamate receptorinteracting protein 1 (GRIP1) as a potential binding partner. GRIP1 is known to bind to the GluR2 subunit of
␣-amino-3-hydroxy-5-methylisoxazole-4-propionate
Physiol Rev • VOL
(AMPA)-type receptors, another type of glutamate receptor in dendrites (196). Immunoprecipitation experiments
showed that GRIP1 bound to KIF5 and the GluR2 subunit
in neurons (Figs. 4 and 5). In addition, when a KIF5
dominant-negative construct lacking the motor domain
was expressed in cultured neurons, the density of GluR2
clusters in dendrites significantly decreased, suggesting
that GluR2 is normally transported to dendrites by KIF5
via its interaction with GRIP1.
Furthermore, when the minimal KIF5-binding domain of GRIP1 was expressed in hippocampal neurons,
KIF5 proteins were recruited to dendrites, while the expression of JIP-3, a well-known binding protein for KLC
(14, 234), led KIF5 proteins to localize predominantly in
axons. These results suggest that the binding of GRIP1 to
KIF5 tends to steer the motor towards dendrites rather
than axons, although the mechanism has yet to be elucidated.
C. KIF5: An mRNA Transporter
GST pulldown assays using the tail domain of KIF5
proteins isolated large RNase-sensitive granules containing hnRNP-U, Pur-␣, and Pur-␤, all of which have been
identified as members of an mRNA/protein (mRNP) complex released from ribosomes following EDTA treatment
(99, 163). This finding suggested that the mRNP complex
is transported by KIF5 proteins (Fig. 4). Further experiments identified various RNA-associated proteins, such as
PSF, DDX1, DDX3, SYNCRIP, FMRPs, and staufen, which
were consistently coimmunoprecipitated by antibodies
against RNA-associated proteins and KIF5 (99). Specific
mRNAs, such as those for CAMKII␣ and activity-regulated
cytoskeleton-associated protein (Arc), but not that for
tubulin, were detected in the same granules, suggesting
the transport of specific mRNAs by KIF5 proteins. Realtime movement of Pur-␣-containing granules in the dendrites of cultured neurons was suppressed by RNA interference (RNAi) of hnRNP-U, Pur-␣, PSF, or staufen, but
not by RNAi of DDX3 or SYNCRIP, suggesting the existence of an essential subpopulation of members of granules for the transport of mRNAs. Proteomic analysis of
these granules has identified at least 42 associated proteins to date. A close examination of interactions among
these proteins will clarify the regulation mechanism of
RNA transport.
The RNP granules were further examined by labeling
different marker proteins. Compatible with the bidirectional movement of granules, cytoplasmic dynein was
detected in the immunoprecipitated fraction, but KLC was
not; this finding is compatible with the fact that the
knockdown of KLC did not affect RNP complex transport
(125).
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An in vitro reconstruction comprising the KIF17cargo membrane fraction, Chlamydomonas flagellum microtubules, KIF17 and ATP, succeeded in moving cargo
vesicles to the plus ends (195). These vesicles have also
been shown to contain NR2B by immunocytochemistry.
These results collectively indicate that KIF17 transports
the NMDA receptor subunit NR2B via an interaction with
the tripartite scaffolding protein complex containing
mLin-10, mLin-2, and mLin-7.
The intracellular function of KIF17 was investigated
using a fluorescence-tagged expression system in cultured neurons. YFP-KIF17 is distributed mainly in the cell
body and dendrites, but not in axons. As YFP-KIF17 colocalized with NR2B, but not with PSD-95, in postsynaptic
region, it was supposed that it was being transported (47).
Functional blocking of KIF17 by the expression of antisense oligonucleotides or using dominant-negative mutants resulted in a significant decrease in the density of
NMDA receptor clusters. When the NMDA receptor was
functionally blocked by the antagonist AP-V, the expression of KIF17 as well as that of NR2B subunits was
upregulated, suggesting the coregulation of KIF17 and
NR2B at the transcriptional level.
The in vivo role of KIF17 was examined in a transgenic mouse overexpressing KIF17, using the calmodulindependent kinase II (CaMKII) promoter (239). The mice
showed significantly better performance in behavioral
tests, such as the Morris water maze tasks for working
memory and spatial memory, suggesting better learning
and memory than wild-type mice. In addition to the increase in the levels of KIF17 and NR2B proteins in the
hippocampus and cerebral cortex, the mRNA expression
levels of both molecules were also high. The upregulation
of NR2B and KIF17 transcription was confirmed by an
increase in the amount of phosphorylated cAMP-response
element-binding protein (CREB). A CREB consensus sequence was found in the promoter regions of both genes.
The enhanced dendritic transport of the NR2B subunit
may stimulate the transcription of NR2B subunit and
KIF17, enhance synaptic transmission, and ultimately induce an improvement in learning and memory in transgenic mice, showing that motor proteins play significant
roles in higher-order brain functions.
D. KIFC2
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A. Transport Between the Endoplasmic Reticulum
and Golgi Apparatus
KIFC2, a member of the C-kinesin or kinesin-14 family, is abundantly expressed in the adult brain (Fig. 2) (57,
148, 185). Although other major C-kinesins, represented
by Kar3 and Ncd, are implicated in cell division, KIFC2 is
supposed to function in membrane transport in postmitotic neurons (185). Immunofluorescence data showed
that KIFC2 was localized to punctate structures in cell
bodies and dendrites. Immunoprecipitation experiments
isolated multivesicular body (mvb)-like organelles, suggesting that KIFC2 is a motor for mvb-like organelles in
dendrites.
E. CHO1/MKLP1
F. KIF21B
KIF21B, a member of the kinesin-4 family, is a plusend-directed motor, highly enriched in dendrites (Fig. 2).
KIF21B has a cluster of negatively charged amino acids
within its stalk domain and seven WD-40 repeats within
its tail domain (128), which is supposed to bind cargoes.
Further characterization of the function of KIF21B is
necessary.
V. CONVENTIONAL TRANSPORT, INCLUDING
ENDOPLASMIC RETICULUM TO GOLGI,
LYSOSOMES, AND ENDOSOMES
Within cell bodies, various membrane organelles
communicate with one another through vesicular transport. To maintain this communication, the recruitment
and integration of membranes to the proper cytoskeletons are essential, processes that are strictly supported
by microtubule- and actin-dependent motor proteins.
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CHO1, or MKLP1, is a member of the kinesin-6 family
(Fig. 2). CHO1 was first identified as a mitotic motor
(194). By transporting the minus end of microtubules
towards the plus end of other microtubules, CHO1 transports microtubules of opposite orientation toward one
another, a process important for spindle elongation during anaphase. During interphase, CHO1 was found to exist
in the dendrites of neurons. Expression of antisense oligonucleotides against CHO1 decreased the population of
microtubules with their minus ends at distal sites in neurites, making the appearance of neurites more axonlike
(199). Whether mixed directionality of microtubules in
dendrites is performed by CHO1 or not will be investigated in the future.
Vesicular structures are budded from the endoplasmic reticulum (ER), which is reticularly distributed
throughout the cell body, and transported towards the
Golgi apparatus, which is concentrated in the perinuclear
region in mammalian cells. This transport is bidirectional,
and the forward transport that is minus-end-directed is
implicated by cytoplasmic dynein. Reverse transport,
from the Golgi to the ER, can be visualized by treatment
with brefeldin A, which only blocks ER-to-Golgi transport
(138). In 1992, kinectin was identified as a binding partner
of KIF5 proteins (225). Although an antibody against kinectin was at first reported to inhibit axonal transport,
isoforms of kinectin expressed in neurons were revealed
to have no specific binding domain for KIF5. The variable
COOH-terminal region of kinectin that binds directly to
the tail region of KIF5 inhibits the microtubule-activated
ATPase activity of KIF5 (169). Thus kinectin may function
as an inhibitor of the transport by KIF5 in nonneuronal
cells.
The major isoform of kinectin, with a molecular mass
of 160 kDa, is concentrated in the ER of nonneuronal cells
and is supposed to function in ER extension (187). However, KIF5 and kinectin seem to be dispensable for ER
extension, since ER structure remains normal in kif5B or
kinectin knockout mice (174, 215). In normal rat kidney
cells, microinjection of an antibody against KIF5 (H1
monoclonal antibody) blocked Golgi-to-ER transport, but
not ER-to-Golgi transport, suggesting that KIF5 is involved in backwards, recycling transport (126). Recent
work with KLCs has characterized the different KLC isoforms bound to purified membrane fractions originating
from the ER and Golgi apparatus (240) (Fig. 4). Indeed,
preincubation of these fractions with GST-fused cargobinding domains of KLC1B and 1D blocked the in vitro
motility of the ER and Golgi fractions, respectively. In
vivo and in vitro assays showed that KIF5B steers ⬃50%
of the transport between these organelles and tends to
bind tubules extended from Golgi following brefeldin A
treatment. Colocalization of KIF5 with organelle-marker
proteins implicated KIF5 in the transport of a substantial
population of cargoes that contained ERGIC58 and p115
but not KDEL receptor or COPs (240).
KIF1C, a member of the kinesin-3 family, is also
reported to participate in this transport (Fig. 2). Under
brefeldin A treatment, the inactive form of KIF1C blocked
Golgi-to-ER transport (32). Disrupted Golgi-to-ER transport was reported neither in kif1C nor in kif5B knockout
mice, suggesting a redundant function of the two motors
in this transport (149, 215) (Fig. 4).
In Xenopus cell lines, Xklp3s, Xenopus homologs of
KIF3 proteins are colocalized with KDEL receptor that is
recycling between the ER and Golgi; thus KIF3s are sup-
1100
B. Lysosomal Transport
Lysosomes are dynamic organelles that move centrally or peripherally, according to a decrease or increase,
respectively, in the pH of the culture medium (63). The
dynamic dispersion of lysosomes during the recovery
phase of subsequent acidification, is blocked by exogenous expression of rigor KIF5 (T93N), a mutant KIF5 that
has no ATPase activity and rigorously attaches to microtubules (151). The same inhibitory effect was also observed in kif5B knockout cells, or in cells expressing
polypeptides corresponding to the kinesin binding domain of kinectin (169, 215) (Fig. 4).
Melanosomes are originally lysosome-related organelles and move dynamically within the cells. Competition between one minus-end-directed motor, cytoplasmic dynein, and two plus-end-directed motors, KIF5B and
KIF3 (kinesin-II), along microtubules, regulates their
movement (45). Because melanosomes arriving at the cell
periphery are tethered to actin filaments by melanophilin,
myosin Va and Rab-27, the effect of plus-end-directed
motors is only visible after perturbation of central aggregation with melatonin, followed by treatment of cells with
MSH, which stimulates dispersion (82, 175, 242). In mammalian melanocytes, antisense oligonucleotides against
KIF5B promote perinuclear aggregation, suggesting an
essential role for KIF5B in pigment dispersion (58). However, in Xenopus cells, the dominant-negative form of
Xklp3, the Xenopus homolog of KIF3, but not a blocking
antibody against KIF5B, inhibits pigment dispersion
(227), which may reflect a difference in functional motors
among species (Fig. 4).
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C. Transport From the Trans-Golgi Network
to the Plasma Membrane
Transport from the trans-Golgi network (TGN) to the
plasma membrane is often compared with dendritic transport in neurons, because in some polarized nonneuronal
cells there is another specific type of transport, similar to
axonal transport in neurons. The default pathway is called
basolateral transport, and the alternative is apical transport. Vesicular stomatitis virus glycoprotein (VSV-G) and
influenza virus hemagglutinin (HA) are representative viral markers of the default and novel pathways, respectively.
KIF13A, a member of the kinesin-3 family, exists as a
dimer in vivo, although recombinant KIF13A expressed in
Escherichia coli was purified as a monomer (147, 148). A
GST pulldown assay using the tail domain of KIF13A
recognized an ear domain of ␤1-adaptin, a subunit of the
AP-1 complex that is engaged in vesicular transport from
the TGN to the plasma membrane. ␥-Adaptin and the
mannose-6-phosphate (M6P) receptor were also coprecipitated from the Triton-X-100-solubilized membrane
fraction using an anti-KIF13A antibody, suggesting a detergent-resistant interaction among them (Figs. 4 and 5).
Overexpression of KIF13A resulted in a redistribution of
the M6P receptor to the cell periphery, suggesting a role
for KIF13A as a transporter of M6P receptor-containing
vesicles via the AP-1 complex.
Besides KIF13A, KIF5 is also recognized as a motor
for transport towards the plasma membrane. Previous
work has shown, however, that colocalization of KIF5 and
VSV-G is observed only transiently, just at the level of the
Golgi, shortly after removal of the perturbation that pools
VSV-G at the ER (126). However, recent reports have
shown a commitment of KIF5 in this transport. Vaccinia
virus A36R membrane protein is reported to bind directly
to the TPR region of KLC and be transported to the
plasma membrane (181). Substrate adhesions in Xenopus
fibroblasts also require KIF5 for keeping their size and
number at the plasma membrane, a process that was
blocked by microinjection of an anti-KIF5 antibody
(SUK-4) or by the expression of a motorless construct
(116). In either case, there was no evidence for where
exactly these cargoes originated from, but the TGN is the
most probable source. The cell body itself is so packed
that it is sometimes difficult to discriminate transport
from the TGN to the plasma membrane, from endosomal
recycling, which is referred to in the next section.
In polarized epithelial cells, an apical transport develops in addition to the default transport starting from
the TGN. In polarized MDCK cells, KIFC3, a member of
the C-kinesin family, transports TGN-derived vesicles
containing annexin XIIIb and HA to the apical plasma
membrane, possibly in cooperation with cytoplasmic dynein (158).
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posed to be the third motor involved in this transport,
although discrepant behavior of marker proteins suggested the existence of multiple cargoes (Fig. 4). KAP3
silencing using an RNAi approach resulted in the fragmentation of the Golgi apparatus and changed the localization
of KDEL receptor, revealing a role for KIF3 proteins in
KDEL receptor-dependent Golgi-to-ER transport (209).
Besides transport, some motor proteins have been
recognized to sustain perinuclear positioning of the Golgi
apparatus. Rabkinesin-6, which was identified as a specific binding partner of the GDP-bound form of Rab-6,
induced the scattering of the Golgi apparatus to the periphery of cells when overexpressed (33) (Fig. 2). When
cytoplasmic dynein or kifc3, a member of the C-kinesin
family, is targeted in mouse, depletion of these minus-enddirected motors also affects the positioning of the Golgi,
suggesting a tug-of-war between these three motors for
the integrity and position of the Golgi apparatus (59, 244)
(Fig. 2).
D. Endosomal Recycling
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which is to enhance the exocytosis of cargoes. Another
member of the kinesin-3 family, KIF1C, is also localized at
podosomes at the plus end of microtubules in the peripheral regions of macrophages, in addition to their distribution in the Golgi region at the centers of cells (113).
In close proximity to the plasma membrane, microtubule motors share vesicle transport mechanisms with
members of the myosin motor family, which move along
the actin network. Among KIFs, there exists a novel kinesin-1 member in Dictyostelium discoideum that was
reported to have an actin-binding region in its tail domain.
The exact function of this region remains to be seen (90).
VI. SLOW AXONAL TRANSPORT
In addition to the fast axonal transport referred to in
previous sections, there exists slow axonal transport in
neurons, which carries cytoskeletal proteins, such as tubulins and neurofilament proteins and glycotic enzymes
at a velocity of 0.1–3 mm/day (219). This form of intracellular transport is reported to be driven by the microtubule-dependent motor proteins KIF5 (220). Regarding the
size of cargoes transporting cytoskeletal proteins, there
has long been a debate between the “oligomer (complexes
of several molecules) hypothesis” and the “polymer (filaments) hypothesis” (16, 197, 220, 245). The majority of
discrepancies have arisen from the differences in the
methods used. Some researchers overexpressed fluorescently labeled proteins in culture systems to investigate
their movement and insist that the fast but infrequent
movement of polymers by fast axonal motors causes only
a small displacement of molecules (21, 235, 236, 248).
However, it remains disputable whether they have reproduced in their culture system slow axonal transport observed in vivo.
On the other hand, others have observed the slow
movement of injected proteins in squid giant axons and
estimated the speed of movement, diffusion coefficients
or diffusion times during displacement, from fluorescence
profiles (39, 40, 220). These investigators used confocal
laser scanning microscopy or fluorescent correlation microscopy and showed that there is a significant difference
in diffusivity characteristics between soluble protein and
tubulin, between tubulin and taxol-stabilized polymerized
tubulin, and between tubulin and neurofilament. In another study, the transport of injected oligomers of neurofilament-M was observed in neurofilament-depleted axons
derived from transgenic mice with neurofilament-H-␤-galactosidase that cannot form filament with neurofilament-M and -L (221). Thus these data propose transport of
neurofilament in the form of oligomers. Collectively, from
these findings, tubulin is observed to be constantly transported in the form of oligomers (40, 197, 220, 221) . This
conclusion seems to be persuasive at present. However,
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Vesicular structures recycle between subcellular
compartments and the plasma membrane. They are at
first endocytosed from the plasma membrane as early
endosomes. Through fission and fusion, they develop into
late endosomes and then into lysosomes for degeneration,
or to recycling endosomes for recurrence at the plasma
membrane through exocytosis. The small GTPases of the
Rab family are known to regulate the sorting of these
membranes through locating different members at different compartments (94). Some Rab proteins have been
revealed to control the association of specific motor proteins with particular endosomes.
Multiple motor proteins have been identified on endosomes (Fig. 4). A tug-of-war between motors driving
cargoes in opposite directions determines the localization
of membranes (7). Which motor proteins are implicated
and regulated by a certain Rab protein seems to depend
on the cell type. In 3T3-L1 adipocytes, early endosomes
containing Rab-5 were reported to be endocytosed by
cytoplasmic dynein, and replacement of Rab-5 with Rab-4
induced KIF3-mediated exocytosis of the endocytosed
vesicles (86); both of these transports by cytoplasmic
dynein and KIF3 were triggered by insulin. Contrarily,
when endosomes are purified from the liver, early endosomes labeled with Rab-4 are driven by KIF5B and KIFC2.
The GDP-bound form of Rab-4 recruits KIFC2 to the
membrane, which keeps endosomes away from the
plasma membrane. When they are transformed to late
endosomes, these vesicles are labeled with Rab-7 accompanied by cytoplasmic dynein and KIF3A, and sometimes
by KIFC2 (8, 9). The KIF3 complex is also detected on late
endosomes and lysosomes in Hela and COS-7 cells (17).
RNAi of KAP3, which binds cargoes to KIF3 tail domain,
or expression of motorless KIF3A, changed the distribution of late endosomes and lysosomes. However, in spite
of this altered distribution, their function, the uptake and
delivery of receptors and ligands through fission and fusion, was not affected by the absence of motor proteins,
suggesting that, with regards to endosomes, the function
of motor proteins just limits the movement of vesicles,
and that the transformation of vesicles rather depends on
Rab proteins.
KIF16B, a member of the kinesin-3 family (148),
binds PIP3-containing vesicles via the PX domain in its tail
region (Figs. 2, 4, and 5). In vivo, KIF16B was localized to
PIP3-positive early endosomes, and this association was
blocked by the persistent GTP-bound form of Rab-5 and
the specific inhibitor of phosphatidylinositol-3-OH kinase
hVPS34 (77). Plus-end-directed motility of KIF16B fixed
endocytosed epidermal growth factors (EGFs) and EGF
receptors beneath the plasma membrane and prevented
them from entering the degenerative pathway. This sustaining function of KIF16B is in contrast to that of KIF3,
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how cytoskeletal molecules are transported by KIF5 remains unknown. Recently, KIF5 was reported to transport
tubulin dimer via collapsing response mediator protein-2
(crmp-2) and KLC (107) (Figs. 4 and 5). The relation of the
identified transport mechanism to slow axonal transport
was not discussed and needs further examination with
regard to dynamics.
VII. POLARIZED SORTING BY
MOTOR PROTEINS
Physiol Rev • VOL
VIII. DEVELOPMENT AND
MOLECULAR MOTORS
To investigate the functions of motor proteins at the
tissue and individual animal levels, molecular genetic approaches have been attempted and have shown that intracellular transport by motor proteins and some motor
proteins with unique functions are critical for many aspects of functions at the tissue and animal levels.
A. KIF3: Left-Right Determination
and Development
In addition to their involvement in axonal transport
and conventional transport, KIF3 heterodimers have a
specific function in intraflagellar transport (IFT). IFT was
first reported as the bidirectional movement of two granulelike particles beneath the flagellar membrane, which is
essential for the assembly of cilia (115). These two particles, A and B, bind two kinds of kinesin-2 motors with
different velocities, kinesin-II (KIF3s, FLA10) and Osm-3,
respectively, in addition to retrograde motors, flagellar
dyneins, resulting in an unusual type of transport in which
particle complexes are conveyed at an intermediate velocity between those of kinesin-II and Osm-3 (170).
kif3A and kif3B knockout mice showed very similar
phenotypes (161, 214). A lack of KIF3A or KIF3B in mice
is embryonic lethal, and embryos on 11.5 dpc show various anomalies, including an abnormal cardiac looping
with equal frequency of L-loop and D-loop, which implies
randomization of left-right determination (161, 214)
(Fig. 7).
At the molecular level, left-right determination can be
detected by the unilateral distribution of marker molecules: a triangular ventral dent in early embryos, called
the “node,” is known to regulate the expression of this
series of genes specifically on the left side. Whole-mount
in situ hybridization of the nodes of kif3B knockout mice
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After molecules arrive at the targeted membrane,
they are specifically incorporated into that membrane in
one of two ways: they are either selectively fused into the
membrane, or they are indiscriminately captured and remain on the membrane by selectively escaping endocytosis (152, 186). Many interactive sequences for specific
recognition have been identified as sorting signals in the
cytosolic domains of targeted molecules (70). Additionally, motor proteins appear to engage in polarized sorting,
presumed from the fact that exogenous expression of
truncated motor domains resulted in their polarized localization, although this could not be influenced by the
cargoes usually bound to the cargo-binding domain in the
COOH-terminal portion of these motor proteins (91, 150).
Thus selective binding to a particular motor limits the
possible destination, because some motor proteins are
restricted to enter either axons or dendrites.
To elucidate the molecular mechanism underlying
polarized sorting by motor proteins, the distribution of
the rigor mutant motor protein was investigated following
infection of differentiated cultured hippocampal neurons
using an adenovirus vector (150). Because the rigor mutant, which lacks ATPase activity, does not move along
microtubules once bound, it remains localized on the
microtubules where it was first recruited. While tailless
KIF5 tended to accumulate at the tip of axon, rigor-KIF5B
(G234A) distributed to a restricted portion of microtubules localized between the cell center and the initial
segment of the axon. The initial segment is known to
work as a diffusion barrier to inhibit backwards leak of
membrane proteins once targeted to the axon (238). Thus
selective binding of KIF5 to microtubules in the initial
segment enables the transport of specific proteins into the
axon. Treatment with low doses of taxol inhibited this
selectivity, suggesting the importance of microtubule dynamics of the initial segment for the polarized sorting
mechanism. In contrast to KIF5, rigor KIF17 was localized
to the initial segment as well as dendrites, reflecting the
distribution of tailless KIF17 in both axons and dendrites
(150).
Interactions between motor proteins and specifically
modified tubulins have been suggested to underlie another polarized sorting mechanism. Some tubulins are
posttranslationally modified within their COOH-terminal
domains, and incorporated into the characteristic microtubules as acetylated, detyrosinated, glycylated, or glutaminated forms. Indeed, perturbation of acetylation and
glutamination affected the transport of KIF5 and KIF1A,
respectively (31, 85, 178).
Observation of neurons during development has also
clarified the existence of multiple mechanisms for axonal
targeting. Even in premature neurons only 2 days in culture, tailless KIF5 selectively accumulated at the tip of a
single neurite (91). However, tailless KIF1A, another axonal transport motor distributed at the tips of all neurites
at this stage, suggested that another sorting mechanism
targets KIF1A to the axon, which is established after the
complete polarization of neurons.
Physiol Rev • VOL
B. Transport of N-Cadherin in Developing Neurons
Conditional knockout of the kap3 gene, encoding an
accessory component of the KIF3 heterotrimer complex
that links the KIF3 tail with the cargo, in mice resulted in
a nonuniform decrease in the expression of KAP3 in the
brain (218). In the cerebral cortex, abnormally hypertrophic regions resembling tumors were distributed in dots
corresponding well to the areas lacking KAP3 expression
(Fig. 7). In these areas, the levels of N-cadherin and
␤-catenin at the cell periphery were markedly decreased.
In an immortalized embryonic fibroblast cell line with a
kap3-null genotype, the release of N-cadherin-GFP from
the Golgi apparatus was significantly impaired, leading to
a decrease in the amount of N-cadherin-GFP at the cellcell boundaries. Indeed, the transport of individual postGolgi organelles containing N-cadherin-GFP showed
winding and unstable outward movements in kap3-null
cells when observed by time-lapse critical angle fluorescence microscopy. These data showed the KIF3 complex
to be a transporter of adhesion molecules such as Ncadherin and ␤-catenin. ␤-Catenin is known to have dual
functions. When ␤-catenin is incorporated into adhesion
junctions, it contributes to cell-cell adhesion, whereas if it
exists in the cytoplasm it tends to get into the nucleus and
act as a transcriptional factor with T-cell factor to enhance cell proliferation, thus causing cancer. Thus KIF3
drives the plasma membrane localization of ␤-catenin,
thereby decreasing the levels of ␤-catenin in the cytoplasm and suppressing tumorigenesis (Fig. 7).
The KIF3 complex was also reported to associate
with APC via KAP3 (92). APC is a known tumor suppressor gene that is involved in the degeneration of ␤-catenin
along with GSK-3␤ and axin. Transport of APC by the
KIF3 motor complex is also supposed to contribute to the
suppression of tumorigenesis.
C. KIF2: A Suppressor of Collateral
Branch Formation
KIF2 is categorized into the kinesin-13 family, members of which have the motor domain in the middle of the
molecule; that is, they are M-kinesins. These kinesins
consist of three separate subfamilies: KIF2A, KIF2B, and
KIF2C/MCAK (Fig. 3). Rather than steering along microtubules with cargoes, KIF2 molecules work as a microtubule depolymerizer. KIF2 steers along microtubules by a
diffusion mechanism to the end of microtubules. Then,
KIF2 decouples tubulin dimers consecutively from the
ends of microtubule filaments (28, 60, 83).
In contrast to the KIF2C subgroup, as represented by
MCAK which functions in mitotic stages (28), KIF2A is
expressed predominantly in the juvenile brain, especially
in neuronal growth cones (159). Kif2A knockout mice
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revealed that the expression of lefty2, the most upstream
gene in the pathway regulating left-right determination,
was already randomized. Scanning electron microscopy
of nodal cells revealed the absence of monocilia in the
nodes of kif3B knockout mice (Fig. 7). Immunocytochemistry showed the localization of KIF3A/KIF3B in the
monocilia in the wild-type nodes (161, 214). Considering
also that the Clamydomonas KIF3 homolog FLA10 is
implicated in IFT of the ciliary components from the base
to the tips of cilia along microtubules (22, 67), it is supposed that cilia are not formed in kif3B knockout mice
because of the absence of KIF3.
To clarify the mechanism underlying the disruption
to left-right asymmetry in kif3B knockout mice, further
work was attempted. When nodal cilia of wild-type mice,
which were thought to be immotile, were observed by
video microscopy, the cilia were unexpectedly rotating
clockwise at ⬃600 cycles/min. Moreover, fluorescent dyelabeled beads added to the extraembryonic fluid in the
node moved from the right to the left. A constant leftward
flow of extraembryonic fluid in the node, named “nodal
flow,” was absent in the nodes of kif3B knockout mice.
These data suggest the possibility that the leftward nodal
flow contributes to the generation of a concentration
gradient of a putative morphogen X towards the left side
of the node for left-right determination.
Leftward nodal flow is observed in other vertebrates
such as rabbits and medaka fish. A video system with a
high temporal resolution enabled a more detailed analysis
of ciliary movements with their axes tilted at an angle of
40° posteriorly (167). Because of this tilt, only a leftward,
but not a rightward, rotation of the cilium would effectively generate hydrodynamic power in a nearly vertical
plane, thereby producing leftward nodal flow.
Membrane parcels labeled with the lipophilic fluorescent dye DiI were observed by confocal microscopy in the
nodes of living embryos; these parcels were released from
the cell surface and the protruding microvilli of the cell
surface, moved rapidly down the stream of the nodal flow,
and were finally fragmented at the location close to the
left wall (216). This transport was suppressed by the
suppression of fibroblast growth factor (FGF) using a
specific inhibitor of the FGF receptor tyrosine kinase,
SU5402, or a dominant-negative recombinant peptide of
an extracellular domain of mouse FGF receptor (FGFRDN). These parcels, named “nodal vesicular parcels”
(NVPs), typically consist of multiple lipophilic granules
and have been found by immunostaining to be associated
with the downstream morphogen candidates, Sonic
hedgehog (SHH) and retinoic acid (RA). These data, originating from research on KIF3 proteins, provide direct
evidence that following an FGF trigger, nodal flow transports NVP-associated morphogens including SHH and RA
toward the left: this is probably a critical phenomenon of
symmetry breaking in mammalian embryos (72).
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D. KIF4: A Regulator of Neuronal Survival
KIF4 is strongly expressed in juvenile neurons where
it localizes in nuclei as well as in growth cones (193).
Several homologs of KIF4, categorized into the kinesin-4
family, have been implicated in chromosome segregation
during the mitotic phase (130). Indeed, in the mitotic
stage, KIF4 was localized to mitotic spindles. Suppression
of KIF4 in neurons using antisense oligonucleotides resulted in the accumulation of L1 within the cell body,
instead of within growth cones, suggesting a role for KIF4
as a transporter of this adhesion molecule for axonal
elongation in neurons (173).
A gene-targeting strategy was applied for the kif4
gene and produced ES cell clones with no KIF4 expression (134). Because kif4 is located on the X chromosome,
single homologous recombination is sufficient to generate
kif4-null cells. Kif4-null neurons that were differentiated
Physiol Rev • VOL
in vitro from the ES cells showed an extraordinarily high
survival rate and were resistant to apoptosis (Fig. 7). GST
pulldown with the tail domain of KIF4 isolated poly-ADP
ribose polymerase-1 (PARP-1) from juvenile mouse brain.
Recombinant KIF4 bound directly to PARP-1 and suppressed its enzymatic activity. Under stable conditions,
the KIF4 and PARP-1 complex was localized in the nucleus. After the membrane was depolarized by high potassium treatment or electrical stimulation, KIF4 was
phosphorylated by CaMKII ␣ dissociated from PARP-1
and released into the cytoplasm, which activated PARP-1
to prevent cell death. In the developing brain, KIF4 by
forming a complex with PARP-1 prevents the protective
action of PARP-1 on cell death (Fig. 7).
IX. REGULATION OF CARGO BINDING
A. Use of an Adaptor/Scaffolding Protein Complex
for Cargo Binding
To transport exact cargoes to the exact destination, a
motor has to discriminate the specific cargo at the starting
point for binding. For that purpose, cargoes usually
present specific adaptors to their motor proteins. As referred to in the previous sections, KIF13A binds to the
AP-1 adaptor protein complex, which then recognizes the
mannose-6-phosphate receptor, enabling its transport
from the TGN to the plasma membrane (Fig. 5) (147).
KIF17 binds to mLin-10, and via this adaptor, it binds to
mLin-2 and the mLin-7 scaffolding protein complex, and
finally to the NMDA receptor NR2B subunit (Fig. 5) (195).
KIF5 binds directly to GRIP1, and via GRIP1, to the GluR2
subunit of the AMPA receptor (Fig. 5) (196). Therefore,
the tail regions of motor molecules recognize and bind to
the adaptor or scaffolding proteins to transport membrane organelles that contain functional membrane proteins. The use of adaptor and scaffolding proteins appears
to be one of the basic mechanisms for the recognition and
transport of cargoes (70, 71, 110).
In addition to adaptor proteins, lipids also contribute
to the specificity of binding. KIF1A and KIF16B specifically bind to phosphatidylinositides, such as PIP2 and
PIP3, respectively, through the PH domains in their tail
regions (Fig. 5) (77, 109). KIF13B binds indirectly to PIP3
via the PH domain of ␣-centaurin (232). However, it remains unknown if this specific binding to the lipid contributes to the selectivity of cargoes or just provides a
reinforcement of their affinity. In fact, in the case of
KIF1A/1B␤, Rab-mediated control of the loading/unloading of the cargo was recently reported (156). The authors
of this study showed that DEN/MADD, a Rab-3GEF (guanine nucleotide exchange factor) directly bound the stalk
domain of the motor and was implicated in the loading of
Rab-3 with synaptic vesicle precursors by producing GTP-
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were born alive, but all died within 1 day (78). Their
brains showed laminary defects in the cortex due to the
delayed migration of cortical neurons (Fig. 7). Visualization of axon bundles with DiI crystals revealed an increased number of horizontally running neurites in mutant mice, whereas axons run longitudinally in wild-type
mice. A more detailed observation of cultured neurons
revealed a significant increase in the lengths of axonal
collateral branches in the knockout mouse, which consequently hindered the forward migration of the cell body
(Fig. 7).
Like KIF2C, KIF2A depolymerizes microtubules in an
ATP-dependent manner in vitro (28, 78). When the behavior of fluorescently labeled microtubules was observed in
the periphery of wild-type cells, elongating microtubules
usually began to depolymerize when reaching the cell
periphery. In contrast, in the absence of KIF2A, microtubules continued to elongate even after reaching the cell
periphery. These data collectively showed that KIF2A is
important for controlling brain wiring by suppressing excessive elongation of branches as a microtubule depolymerizer, which in turn is essential for the migration of
neurons.
Recent work searching for the functional complex
containing KIF2A has revealed that KIF2A directly interacts with phosphatidylinositol 4-phosphate 5-kinase ␤
(PIP5K␤), which is implicated in the endocytosis of the
plasma membrane through the production of PIP2 (157).
In addition to the coaccumulation of these two proteins at
the tips of developing neurites in vivo, PIP5K␤ was shown
to increase the microtubule depolymerizing activity of
KIF2A in in vitro assays. The cooperative function of
PIP5K␤ and KIF2A was proposed to be a fundamental
mechanism for the proper elongation of neurites through
the coupling of actin and microtubule networks.
B. Autoinhibition/Phosphorylation
When KIF5s exist in the cytoplasm and do not bind to
cargoes, they are in a folded conformation (38, 51, 68, 87,
211) . Two flexible domains in the stalk region enable an
interaction between the tail region and the neck/hinge
region localized near the motor domain. For this folding
to occur, the presence of five COOH-terminal amino acids
is critical to keep KIF5 in the inhibitory folded state; that
is, in a low binding state for microtubules or in a low
ATPase activity state, in which KIF5 is not activated by
microtubules (23, 108). Other reports have also shown the
critical role of other tail globular regions for folding (252).
To induce the folded conformation, KLC is also essential
(233). Autoinhibition is overcome by the binding of cargo
to KIF5s.
In the case of KIF1A, which exists as a monomer in
the cytosol, an intramolecular interaction between the
FHA domain and the following coiled-coil domain 2 was
reported to inhibit microtubule binding activity as well as
multimerization (121). Disruption of this interaction by
point mutation, deletion, or hinge-removing mutation enhanced the transport and accumulation of KIF1A at the
distal region of neurites. An autoinhibition mechanism
involving the tail and hinge domains has also been reported for Osm-3, a KIF3 homolog (87), and for MCAK/
KIF2C (140).
Recent work has revealed, however, that autoinhibition is not necessarily overcome by binding to cargoes
(240). The authors of this work showed that additional
binding of deleted KLC fragment containing only KIF5binding domain to the cargo-bound KIF5 complex could
inhibit the motility of KIF5 motors. They denied the possibility that newly formed unbound KIF5 complexes in the
Physiol Rev • VOL
cytoplasm were involved in this inhibition, because depletion of a motor complex in the cytoplasm did not affect
the results. As KIF5 forms a dimer with two binding sites
for KLC and the tail domains, an autoinhibition mechanism may doubly regulate the activity of KIF5 proteins. A
recent report proposed a dual mechanism for autoinhibition, in which the cooperative binding of FEZ1 and JIP1 to
the tail globular domains of KIF5 and KLC, respectively,
released the KIF5 complex from autoinhibition (12).
FEZ1/UNC-76 binds directly to the tail globular domain,
which is in the COOH terminus of the light chain-binding
and the cargo-binding domains of KIF5. Different from
other cargo molecules, FEZ1 does not accumulate in the
axon, when KIF5 is depleted and traffic jams of untransported cargoes form in C. elegans (42). Considering that
this molecule is phosphorylated downstream of a specific
kinase, it may function as a regulator of the motor protein
rather than the cargo adaptor.
Arriving at the destination, cargoes must be released
from the motor proteins before being introduced into the
targeted membrane or any other destination. Previous
work showed that the phosphorylation of KIF5 regulates
the binding affinity of cargo vesicles (122, 188). Recently
the precise mechanism was found as follows: phosphorylation of KLC by GSK-3␤ detaches KIF5 from transported
cargoes without changing the ATPase activity or microtubule affinity of KIF5 (142, 143). Moreover, recent work
with KIF17 clarified that the CAMKII-dependent phosphorylation of KIF17 controlled the interaction between
KIF17 and its direct binding partner Mint1/mLIN-10 in
vitro as well as in vivo (48). The authors of this study
identified the phosphorylated Ser, Ser-1029, at the COOHterminal tail domain of KIF17, and visualized the phosphorylation-dependent transport and detachment of the
cargo by immuocytochemistry and FRET, respectively.
Contrarily, the affinity for microtubules or the motility of KIF5 proteins is reported to be regulated by the
phosphorylation of binding proteins. FRET analysis detected the dissociation of KIF5B from ␤-tubulin III following the phosphorylation of JNK proteins within the axon
(208). Collectively, phosphorylations of the motor complex and adaptor protein seem to regulate the unloading
and transport efficiencies of the cargo, respectively. However, more data are needed to generalize this rule. In the
case of the mitotic motor MCAK, phosphorylation of the
motor protein was reported to change its localization and
ATPase activity, or localization only, depending on the
phosphorylated sites (2, 164).
Heat shock protein 70, a representative scaffolding
protein, has also been implicated in interaction between
KIF5 and cargo vesicles (226). The molecular mechanism
is still unknown, but this reaction occurs in a MgATPdependent and N-ethylmaleimide-sensitive manner, which
may explain the two- to threefold increase in the yield of
motor-associated vesicle population when brain samples
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rab3 from the GDP-form, which started the transport and
GTP-Rab-3, in turn, was supposed to be released by GTPase activating protein (GAP) at the vesicle’s destination,
the synaptic terminals. Considering that KIF16B was also
reported to be displaced from the cargo in the presence of
dominant-negative Rab-5(77), these data may presume a
new regulation system for lipid-motor interactions.
Some adaptor proteins are supposed to change the
processivity of bound motor proteins along microtubules.
Calsyntenin-1 was shown to bind directly to KLC by a
yeast two-hybrid system and immunoprecipitation (112).
A point mutation in the binding site for KLC did not
change the speed of transported vesicles, but instead,
tended to increase the cargo fraction that is static or
moves retrogradely. Drosophila enabled was also reported to bind KIF5 directly and reduce its transport
activity under the control of Abl tyrosine kinase (129).
The precise molecular mechanism involved will be clarified by the further experiments.
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are dissociated in buffer containing EDTA or N-ethylmaleimide.
After releasing their cargoes, motor proteins are supposed to be degraded at the destination, since little retrograde transport of anterograde motors has been detected within axons for reuse (69). An unknown mechanism including phosphorylation and degradation may
function to clear up dissociated motor proteins.
C. Tug-of-War Between Motor Proteins
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X. STRUCTURE OF MOTOR PROTEINS
A. Structure of KIFs and Microtubules
In 1989, molecular structure of KIF5 was revealed to
be composed of the globular motor domain, the stalk
domain, and the tail domain (68, 190), and KLC was
shown to bind the tail domain by immunoelectron micrographs decorated with an anti-KLC antibody (68). In 1995,
three independent works clarified the three-dimensional
structure at medium resolution (⬃30 Å) of microtubuleKIF complexes: microtubule-KIF5 and microtubule-Ncd,
through computational analysis of cryo-electron micrographs (cryo-EM) (74, 76, 104). The authors of these
studies not only showed that a motor domain of KIF5 is
oriented to an identical region of a single protofilament,
with an interval of 8 nm, but also clarified that Ncd, a
C-kinesin, is oriented to the identical region to KIF5,
irrespective of its original opposite directionality. In 1996,
atomic structures of the motor domains of KIF5 and Ncd
in the ADP-bound state were first solved by X-ray crystallography. Contrary to expectations, they revealed a structural similarity to myosin, although the amino acid sequences are apparently different between KIFs and myosin, and they show opposite kinetics to interact with
microtubule or actin protofilament during the ATP hydrolysis cycles (117, 184). While KIFs are in the weak binding
state when bound to ADP, myosin binds strongly to actin
filament in the ADP-bound state. As deduced from the
information about the cryo-EM and mutational studies,
the core domains of microtubule interacting-interphase
were identified as loop7/8, loop11, ␣4, and loop12/␣5 (105,
165, 253).
After these works, several crystal structures of kinesin in the different subfamilies (kinesin-1, -3, -5, -13, and
-14) have been solved, revealing that they have common
structures of the catalytic core (106, 114, 117, 155, 162,
184, 204, 228, 253). Some differences in the flexible loop
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Anterograde axonal transport is conducted by various kinds of KIF, while cytoplasmic dynein is the only
motor presently known to drive retrograde axonal transport. When real transport is observed in living axons,
cargo vesicles do not necessarily move one-way. They are
often carried back and forth within the axon and are
sometimes stationary. This phenomenon is due to a tugof-war between anterograde and retrograde motors, and
the net result is that they are transported towards their
own destination (145). Indeed, motor proteins of opposite
directionality have been recognized as being attached to
the same vesicles at the same time. Direct interactions
between subunits of the dynactin complex and those of
KIF complexes have also been reported; for example, an
interaction between Glued p150 and KAP3 (26), or another between dynein intermediate chain and KLC (124).
During movement in one direction, one motor must be
turned on and the other off. How they are activated and
inactivated alternatively remains unknown.
Some motors play multiple roles in transporting different kinds of cargoes. They sometimes have to choose
different linking forms to bind appropriate cargoes, such
as directly, via scaffolding/adaptor proteins and lipid.
Among exogenously expressed cargoes, competitive interaction for a motor protein was observed. For example,
Alcadein and JIP-1 are binding proteins for KLC and compete for KLC. More Alcadein was reported to be dissociated from KLC-KIF5 complex as the expression of JIP-1
increased (3). How a motor protein judges the order of
priority of different cargoes and how it switches to another linking form for the appropriate cargoes still remain
unclear. Moreover, in contrast to small cargoes like protein complexes, the size of vesicular cargoes is at most 10
times larger than that of motor proteins (Fig. 1). Vesicular
membranes are supposed to contain other membrane
proteins and lipids in addition to identified cargo proteins
directly bound to the motor complexes. Whether motor
proteins recognize such accompanied molecules and subsequently regulate their transport also needs to be examined in the future.
Fusion and fission of fluorescently labeled transported vesicles are commonly observed by time-lapse
video microscopy. How motor proteins adapt themselves
to a specific certain type of vesicle needs to be examined.
It is also unknown how much a single motor protein
contributes to driving its cargo along microtubules,
whether it continues to bind cargo all the way to the
destination or if it shifts to another motor and detaches
from the cargo. Electron microscopy has revealed multiple crossbridges between cargo vesicles and microtubules (Fig. 1). Increasing data have also suggested cooperation among multiple motors in the transport of vesicles
(11, 123). Together, biophysical analysis of the movement
of cargoes and theoretical studies suggest that one to
three anterograde motors on average simultaneously
steer one vesicle in vivo. Further studies are needed to
solve the regulatory mechanism controlling binding and
unbinding of cargo, and motor activity.
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B. Mechanism That Couples ATP Hydrolysis
and Conformational Change
Similar to G proteins and myosins, kinesin holds
nucleotide in the nucleotide binding pocket formed by
well-conserved P-loop (amino acids 97-105 in KIF1A). An
adjacent loop L9 (amino acids 199-218), which is called
switch I from the analogy to G proteins, changes its
conformation during the ATPase cycle, which is considered to trigger the hydrolysis reaction as well as the
release of the product phosphate and the release and
entry of nucleotide (Fig. 6). From the analogy to myosin,
where nucleotide enters and leaves from one end of the
nucleotide binding pocket and phosphate leaves from the
other side, the conformational change related to the phosphate release is called as the opening of the “back door.”
Recent structural studies have revealed details of this
conformational change (155). Similarly, release of ADP
and entry of ATP would take place through the “front
door.” However, atomic structures of these states are
currently missing, and little is known about the structural
details of this process.
Physiol Rev • VOL
FIG. 6. Schematic model of the molecular structures of KIFs to
steer along a protofilament. While KIF1A slides along a protofilament by
using electrostatic interactions, KIF5 directs another head towards the
plus end by a hand-over-hand mechanism.
The hydrolysis reaction is tightly coupled to the conformational change of the switch I loop L9. How is this
chemical cycle coupled to the mechanical cycle? The
microtubule binding surface of kinesin is mainly composed of a series of structural elements loop L11-helix
␣4-loop L12-helix ␣5 (amino acids 248 –324), which is
called as switch II complex from the analogy to G proteins. Crystal structure of KIFs revealed that this complex
takes two stable conformations: “up” and “down.”
In the “up” conformation, helix ␣4 protrudes on the
surface so that it fits into the intradimer groove between
␣- and ␤-tubulins. Loop L11 extends toward tubulin,
which contributes to the strong binding to microtubules.
This is the “up” conformation; KIFs can bind to microtubules strongly (strong binding state).
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regions presenting the difference of the length of the
loops or different electric charges are expected to explain
the diversity of the functions among kinesins of different
subfamilies.
On the other hand, the atomic structure of tubulin
was first reported by Nogales et al. in 1998 (160). Although
this structure was solved using nonphysiological zincinduced tubulin sheet, this work was the first report of the
atomic structure of tubulin in the straight microtubule
protofilament. In 2000, crystal structure of tubulin taking
the curved conformation was solved in the form complexed with stathmin, which is one of the microtubuledestabilizing MAPs (41). These two tubulin structures
taking the straight and curved conformation are thought
to represent the stabilized and destabilized form of microtubule, respectively. However, molecular mechanism
of microtubule-dynamics is still unclear, and future studies are needed.
In the current stage, because of the lower resolution
of the cryo-EM structure, in silico docking of the crystal
structures of kinesin and tubulin to the medium-resolution cryo-EM structure of microtubule is the mainstream
to construct the pseudo-atomic model of kinesin-microtubule complex. Recently, cryo-EM structures of KIFmicrotubule complex have been obtained at much higher
resolutions (9 –12 Å) (73, 103, 203), and this technique is
expected to have the potential ability to achieve the nearatomic resolution of kinesin-microtubule complex in the
near future. Crystal structure of KIF-tubulin complex is
also another potential candidate to reveal the real atomic
structure of kinesin-microtubule complex.
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C. Processive Movement of KIF5
KIF5 is a highly processive motor, which hydrolyzes
more than 100 molecules of ATP and translocates along
microtubules successively before detaching from it (50,
80). Because monomeric constructs detach from microtubules after each cycle, dimerization is essential to move
successively along microtubules for KIF5 (55, 56).
The “walking” model or a “hand-over-hand” model is
now a widely accepted model of processive movement
(Fig. 6) (249). In other words, a motor needs two legs, as
we do in walking. When one foot steps forward, the other
foot needs to be firmly attached to the rail for a motor to
be able to “walk” without ever detaching from the rail.
However, there has been a long controversy about the
symmetricity of the walk, since the dimeric kinesin must
walk along a protofilament with two identical right feet,
as the structural data show. The “inchworm” model, that
is, one head always leads the other head and moves like
an inchworm, and the “asymmetrical hand-over-hand”
mechanism, a dimeric motor translocates along a protofilament with alternately different step size, like limping,
have been proposed (4, 81).
The first evidence for a hand-over-hand model was
obtained from the experiment measuring the steps of
single kinesin head labeled with Cy3 fluorescence, which
presents the step size of 17.3 ⫾ 3.3 nm, corresponding to
the two steps of kinesin (250). Further evidence was then
acquired from the conformational change of the neckPhysiol Rev • VOL
linker synchronized with ATP hydrolysis (180). By adding
electron paramagnetic resonance or a gold cluster tag to
cysteine-333 in the necklinker, its movement during the
ATP hydrolysis cycle was observed in both wild-type KIF5
as well as a low ATPase mutant construct. These data
suggest that the necklinker extends towards the plus end
while binding to ATP (Fig. 6), and then becomes mobile
simultaneously with the release of ␥-phosphate after ATP
hydrolysis.
The neck region of KIF motors consists of two domains: a necklinker that attaches to the catalytic core, and
a coiled coil region that unites two motors for dimerization (Fig. 6). Experiments inducing deletion or crosslinking into these regions clarified that a release of necklinker
from the catalytic core is necessary for the processivity of
motor proteins (182, 224). On the other hand, inhibition of
the unwinding of the coiled coil region did not disrupt
motility, although the overall length of two necklinkers is
not sufficient for covering the step size of the motor.
Instead of an unwinding effect, the positive charge of this
region, which interacts electrostatically with the negatively charged COOH-terminal region of tubulin, rather
contributes to the processivity of the motor (222, 224).
Because monomeric KIF1A motor mainly uses this electrostatic mechanism to sustain its association to microtubules when in the weak binding state (165, 166), it is
surprising that dimeric motor needs the same system,
although either head is always connected to microtubules
and protected from detachment. Recent work using single
molecule FRET succeeded in examining the intermediate
conformation of the heads between steps (144). At lowlimiting concentration of ATP, the dimeric kinesin was in
one-head bound state, where the detached head leaded
another head attached to the microtubules (Fig. 6).
The mechanochemical coupling has also been investigated by a biophysics assay. To move one-directionally
along a protofilament, two heads of dimeric KIF5 have to
behave differently but coordinately depending on their
relative positions. This “gating” mechanism was observed
in both heads (13). ATP hydrolysis of the first head tightens the binding of the second front head to microtubules,
which then accelerates the detachment of the first head
for forward displacement steered by the power-stroke of
second head (35, 55). The gating mechanism is supposed
to be mediated by the reciprocal mechanical strain
through the direct or indirect connection of the heads
via the necklinker and protofilament (13). How the strain
then affects the binding and hydrolysis will be clarified in
the future.
D. Structure of C-Kinesins
In addition to the fundamental mechanism common
to KIF5, other KIFs play specific roles in vivo by using
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In the “down” conformation, helix ␣4 is embedded
between the groove by helix ␣5 and helix ␣6 so that the
microtubule binding surface of kinesin become smooth
and does not fit to the groove between the dimer. Loop
L11 is wound up to helix ␣4 so that it can no longer
interact with tubulin. In this conformation, KIFs do not
strongly bind microtubules (weak binding state).
Crystal structures by Nitta et al. (155) suggested that
the conformational change of switch II complex is tightly
coupled to that of switch I through formation and breakage of salt bridges between switch I and II. The “back
door,” a key interaction for hydrolysis and phosphate
release, is formed by the well-conserved salt bridge between switch I and switch II. Thus the formation and the
breakage, namely, closure and opening, of the “back
door” couples the hydrolysis reaction to the conformational change of the microtubule surface from “up” to
“down” (strong binding to weak binding).
Upon the release of ADP and the following ATP
binding, the reverse conformational change is expected to
occur, from “down” to “up.” This conformational change
would be coupled to the conformational change of switch
I (“front door”). However, structural details are yet unclear due to the lack of the atomic structures of this state.
E. Monomeric Motor KIF1A: How Does It Move?
In contrast to other motor proteins, such as KIF5,
Ncd, and myosin, KIF1A exists as a monomer in solution
(168). Therefore, it cannot move processively by the
“hand-over-hand” mechanism (56). However, the shortest
motor domain construct of the KIF1A molecule, C351,
moved along a microtubule processively over a disPhysiol Rev • VOL
tance of more than 1 ␮m (166). On average, C351
hydrolyzed nearly 700 ATP molecules before detaching
from the microtubule, which is more than 7 times larger
than KIF5.
In contrast to KIF5, however, the movement of single
C351 molecules along the microtubule was not smooth
and actually appeared to be oscillatory. Quantitative analysis of this apparently oscillatory movement revealed a
linear increase in mean displacement and mean variance,
indicating a unidirectional constant-velocity movement
accompanied by Brownian movement (5). When comparing the movement of C351 with that of K381, a dimeric
construct of a conventional KIF5 molecule, plots of mean
square displacement against time {[MSD, ␴(t)]} from C351
fit well with biased Brownian movement (166).
The presence of the diffusional component in the
movement of KIF1A suggests that KIF1A is loosely attached to microtubules even in the state in which other
KIFs are detached from microtubules. Thus a singleheaded KIF1A would be able to advance without fully
detaching from microtubules. Indeed, a monomeric
KIF1A construct C351 had higher affinity to microtubule
even in the ADP state, when other KIFs are in the weakbinding state. C351 in ADP state, however, was not stably
bound to microtubules but showed a one-dimensional
Brownian movement along the microtubules (166).
In search for the loose anchor for KIF1A, a classspecific extension in the loop L12 was noticed. This extension contains five successive lysine residues so that it
is named “K-loop.” This highly positively charged loop
extends toward microtubules in the ADP state. The Kloop is supposed to interact with the negatively charged
domain of tubulin COOH-terminal named the “E hook,”
which contains many glutamates (165, 166). Mutation
constructs with fewer lysines in the K-loop produced a
significantly decreased processivity of the motor (165),
suggesting that the positive charge of the K-loop contributes to the affinity of KIF1A for microtubules.
F. Structure of KIF2C/MCAK: A Common
Mechanism for Microtubule Destabilization
KIF2 depolymerizes microtubules at the ends of microtubules. Within cells, however, KIF2 does not necessarily exist only at microtubule tips, but usually binds to
the sides of microtubules and moves rapidly towards the
ends by diffusion through an interaction between the
positively charged neck and negatively charged E-hook of
tubulin (83). However, in contrast to KIF1A, this onedimensional diffusion has no directional predominance
and does not require the hydrolysis of ATP (28, 60, 83).
The deletion of the positively charged residues in the neck
domain of KIF2C reduces the microtubule depolymerizing
activity of KIF2C, which is then rescued by the substitu-
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their specifically modified structures. In the following
chapters, three examples, Ncd, KIF1A, and KIF2C, are
discussed.
C-kinesins, which have a motor domain in the COOH
terminus of the molecules, drive minus-end-directed motility. Because of their opposite directionality to other
N-kinesins, they have been studied for the mechanism
determining the directionality of motors. The three-dimensional structure of the Ncd-microtubule complex was
first reported in 1995, along with KIF5 (74, 75, 184, 207).
Despite opposite directionality, the catalytic core of Ncd
binds to the identical site in the microtubule to KIF5.
To determine which domain is essential for directionality, replacement of a domain of KIF5 with a corresponding Ncd domain, or induction of a point mutation within
the region concerned, has been performed, showing that
directional bias is determined by the neck/core interaction but not by the catalytic core itself (20, 34, 61, 183).
Indeed, a mutant Ncd with a single amino acid change in
the neck region presented reverse motility, which became
then bidirectional by inducing another mutation in the
facing catalytic core (34). When a three-dimensional
structure of dimeric Ncd with microtubules was solved by
cryo-EM and computational analysis, the unbound second
head tilted towards the minus end, while the unbound
KIF5 head oriented to the plus end (75), reflecting their
original directionality.
Analysis of the crystal structure of Ncd revealed that
the neck region of Ncd does not have a necklinker like
KIF5, but that it consists of a solid ␣-helix, although the
catalytic core of Ncd is located in the similar orientation
to microtubules to that of KIF5 (183). Moreover, displacement of Ncd along microtubules seems biased clockwise,
while directed towards the minus end (34). Molecular
biophysics experiments have shown that Ncd has a mechanochemical cycle that is totally different from that of
KIF5 (27). Ncd does not move in consecutive 8-nm steps
like KIF5, but, on average, about a 9-nm large axial displacement synchronized with ATP capture and tends to
detach from the microtubules, showing the characteristics of a nonprocessive motor (27, 37). Thus some drastic
difference between Ncd and KIF5 may be derived from
the different arrangement of the neck. To obtain the
details by biophysiological data on its motility, discovery
of a processive type of C-kinesin motor is awaited.
1109
1110
TABLE
1.
Cargo complex transported by KIFs
KIF
Form of Transport
Cargo Molecules
Binding Protein Complex
Reference Nos.
Axonal transport
KIF1A
Synaptic vesicle precursors
Direct
KIF1B␤
KIF1B␣
KIF5A
KIF5
Osm-3
KIF13B
KLC
Direct
Direct
Direct
Direct
Direct
Direct
Direct
KLC
KLC
KLC
KLC
KLC
Direct
Direct
KAP3
KAP3
KAP3
KAP3
KAP3
Neurofilament
Tubulin dimer
Mitochondria
Mitochondria
Crmp2
Milton
Syntabulin
GAP43, synapsin
Syntabulin, syntaxin
SNAP25
DISC1, NUDEL,LIS1,14-3-3␧
␤-Dystrophin
Neurofibromin
Huntingtin-associated protein1
Torsin A
JIPs, ApoER2
APP
Alcadein
Enabled
FEZ1
Complex A
Fodrin
APC, Par3
N-cadherin, ␤-catenin
Choline acetyltransferase, acetylcholinesterase
IFT complex
Kv channel
IFT complex
Complex B
Centaurin-␣1
PIP3
Discs large tumor suppressor protein
L1
Direct
Direct
KIF4
168
109
202
254
153, 241
139
243
107
44
19
36
212
29
217
127
52
132
98
234
95, 97
3
129
12
22, 170
213
154, 200
218
10
46
170
232
79
54
173
Dendritic transport
KIF17
KIF5
Direct
Direct
Direct
KIFC2
CHO1/MKLP1
KIF21B
Lin-10, Lin-2, Lin-7, NR2B
GRIP1, GluR2
hnRNP-U, Pur-␣, Pur-␤
mRNP complex
Multivesicular bodies
Minus-end microtubule
195
196
99, 163
185
194
128
Conventional transport
KIF5
Direct
KIF1C
KIF3A/3BXKPL3
KAP3
Rabkinesin-6
KIFC3
KIF13A
KIFC2
KIF16B
KIF4
Direct
Direct
Direct
ER
Golgi-to-ER
Lysosomes
Melanosomes
Kinectin
ERGIC58, p115
Endosomes
Golgi-to-ER
Podosomes
Golgi-to-ER
Lysosomes
Melanosomes
Early endosomes
Late endosomes
Golgi
Golgi
TGN-to-plasma membrane
TGN-to-plasma membrane
Early endosomes
Early endosomes
Vaccinia virus A36R
Substrate adhesion
Rab4
KDEL receptor
Rab4
Rab7
Rab6
Annexin-XIIIb, HA
AP-1 (␤-adaptin), M6P receptor
Rab4
PIP3, EGF, EGF receptor, Rab5
PARP-1
187
126, 240
151
45
181
116
8, 9
32
113
209
17
227
86
8, 9
33
244
158
147
8, 9
77
134
KIF, kinesin superfamily protein; KLC, kinesin light chain; KAP3, kinesin superfamily-associated protein 3; IFT, intraflagellar transport.
Physiol Rev • VOL
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KIF3A/3B
Synaptic vesicle precursors
Mitochondria
Synaptophysin, synaptotagmin, Rab3A
PIP2
Liprin-␣
Synaptophysin, synaptotagmin, SV2
KBP
PSD-95, PSD-97, S-SCAM
tion of this neck with K-loop of KIF1A (171). Thus the
electric interaction with the negatively charged E-hook of
tubulin seems generally used by KIFs to sustain its affinity
for microtubules. Upon arriving at the ends, KIF2C/MCAK
hydrolyzes multiple ATPs and removes, on average, ⬃20
tubulin dimers before finally dissociating from the end.
This characteristic behavior of KIF2 seems to be derived
from its molecular structure as an M-kinesin. The interaction of KIF2 with microtubules is expected to provide
another structural hint regarding the function of KIFs.
1111
The crystal structures of KIF2C in the ADP- and
AMP-PNP-bound states were investigated to reveal the
molecular mechanism underlying microtubule depolymerization (162, 204). The crystallized monomeric construct includes a neck region that is located NH2-terminal
to the motor domain in the M-kinesin. In contrast to other
KIFs, KIF2C shows a particular conformation as an Mkinesin (162). A neck region of KIF2C forms an ␣-helixrich structure extending vertically downward to the microtubules. The loop2 domain of KIF2C protrudes like a
Physiol Rev • VOL
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FIG. 7. Intracellular transport is essential for the proper function of living
organelles at the cellular, tissue, and individual animal levels. [Data from Hirokawa and co-workers (78, 134, 161, 218,
251, 254).]
1112
indispensable. Accumulating evidence has just begun to
reveal an elaborate mechanism for the recognition and
regulation of intracellular transport (sect. IX). Third, the
intramolecular mechanism underlying the conformational
change in motor proteins is also discussed, through analysis of the crystal structures and the cryo-EM images of
motor proteins (sect. X).
The intracellular “physical distribution system” sets
up widespread networks within cells and manages proper
execution of totally balanced transport. We still do not
understand very well the mechanism involved. There are
still many KIFs whose functions are not clarified. We do
not yet fully know how motor molecules recognize and
bind to their cargoes, how they choose a particular transport route and, finally, how they identify the destinations
for their cargoes. Besides, in addition to KIFs, myosin
superfamily proteins and dynein superfamily proteins are
also involved in intracellular transport (65, 229). It is still
unknown how they recognize one another and how, on
certain occasions, they take over the cargoes or cooperate to accomplish this task. As for a supervision mechanism to coordinate transport by multiple motors, we have
no knowledge at all. Further experiments will reveal the
detailed mechanism of intracellular transport in the future.
ACKNOWLEDGMENTS
XI. CONCLUSIONS AND
FUTURE PERSPECTIVES
Cells use various types of KIF motor, including monomeric-type, dimeric-type, heterodimeric-type, NH2-terminal motor domain-type, middle motor domain-type, and
COOH-terminal motor domain-type motors (Figs. 2 and
3). Such motors transport various molecules in the form
of membrane vesicles, protein complexes, and mRNAprotein complexes to their proper destination, at a proper
velocity (Table 1). In this review, each type of molecular
transport implicated by KIF motors has been described
first in terms of the route taken, the particular cargoes
being transported, and the particular motor proteins involved (sects. III-VII) (Fig. 4). In many routes, multiple
motor proteins play complementary, redundant parts to
accomplish the transport process within cells. At a higher
level, such as in tissues and individual animals, motor
proteins have been implicated in determining cell morphology, cell-cell contacts, and differentiation (Fig. 7).
KIFs even control important events such as left-right axis
determination during development, brain wiring, activitydependent neuronal survival, suppression of tumor formation, and higher cortical functions, such as learning
(sect. VIII).
Second, interactions between molecules are mentioned. To execute multistage transport processes, the
intermolecular interaction between motor and cargo is
Physiol Rev • VOL
We are grateful to our co-workers for providing experimental data, valuable discussions, and help.
Address for reprint and other correspondence: N. Hirokawa, Dept. of Cell Biology and Anatomy, Graduate School of
Medicine, Univ. of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 1130033, Japan (e-mail: [email protected]).
GRANTS
This work was supported by a Grant-in-Aid for Specially
Promoted Research from the Ministry of Education, Culture,
Sports, Science, and Technology of Japan (to N. Hirokawa).
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