REVIEW
Rab3A small GTP-binding protein in Ca2+-dependent
exocytosis
Yoshimi Takai1,2,*, Takuya Sasaki1, Hiromichi Shirataki1 and Hiroyuki Nakanishi2
1
2
Department of Molecular Biology and Biochemistry, Osaka University Medical School, Suita 565; and
Takai Biotimer Project, ERATO, Research Development Corporation of Japan, Kobe 651-22, Japan
There exists a small GTP-binding protein (G protein) superfamily, consisting of more than 50
members, from yeast to mammal. The Rab family belongs to this superfamily and is implicated in
intracellular vesicle trafficking. Rab3A small G protein is a member of the Rab3 subfamily which
belongs to this Rab family. The regulators and downstream targets of Rab3A have been isolated,
and evidence is accumulating that Rab3A and these Rab3A-interacting proteins are involved in
Ca2+-dependent exocytosis, particularly in neurotransmitter release from nerve terminals.
Introduction
There is a small GTP-binding protein (G protein)
superfamily, consisting of more than 50 members, from
yeast to mammal, as shown in Table 1. According to
their structures, these small G proteins are grouped into
at least six families: the Ras, Rho, Rab, Arf, Sar1 and
Ran families. The Ras subfamily of the Ras family,
consisting of Ha-Ras, Ki-Ras and N-Ras, regulates
gene expression at least through a MAP kinase cascade.
The Rho family regulates cytoskeleton through reorganization of actin filaments and gene expression through
another MAP kinase cascade, a JNK kinase cascade. The
Rab family regulates intracellular vesicle trafficking. The
Arf and Sar1 families also regulate intracellular vesicle
trafficking. The Ran family regulates protein transport
into the nucleus. It is therefore well established that
small G proteins play an important role in various cell
functions.
Transmembrane proteins are transported from one
membrane compartment to another by vesicles. For
instance, membrane receptors such as the EGF and
PDGF receptors are synthesized on ribosomes and
transported to the endoplasmic reticulum (ER) membrane, from where they are transported to the plasma
membrane through the Golgi complex by vesicles.
Soluble substances, such as those secreted outside the
* Corresponding author: Fax: +81 6 879 3419.
# Blackwell Science Limited
cell from the plasma membrane, are also transported by
vesicles. For instance, hormones and digestive enzymes
are synthesized on ribosomes and transported into the
ER lumen from where they are transported to the
plasma membrane through the Golgi complex by
vesicles and finally secreted from the cell. Exocytosis,
endocytosis and transcytosis are performed by intracellular vesicle trafficking. There are two exocytotic
pathways: one is a regulated pathway and the other is
a constitutive pathway. In the former pathway, in most
cases exocytosis is regulated by Ca2+. Vesicle trafficking
is also involved in various other cell functions, such as
the formation of cell polarity, cytokinesis and cell
motility.
There are at least four principal mechanisms in
intracellular vesicle trafficking, as shown in Fig. 1:
(i) budding of a vesicle from the donor membrane;
(ii) targeting of the vesicle to the acceptor membrane; (iii) docking of the vesicle to the acceptor
membrane; and (iv) fusion of the vesicle with the
acceptor membrane. The evidence that the Rab family
members regulate intracellular vesicle trafficking was
first obtained genetically in the budding yeast Saccharomyces cerevisiae. In the yeast, vesicles are transported to
a bud to enlarge the plasma membrane as well as to
secrete many proteins outside the cell. Many genes
essential for secretion were isolated and named the Sec
genes (Novick et al. 1980). One of the Sec genes,
named the Sec4 gene, was shown to encode a small G
Genes to Cells (1996) 1, 615–632 615
Y Takai et al.
Table 1 Small G protein superfamily
Ras
Rho
Rab
Ha-Ras
Ki-Ras
N-Ras
R-Ras
RaI
Rap1A
Rap1B
Rap2
Tc21
RhoA
RhoB
RhoC
RhoG
Rac1
Rac2
Cdc42
Tc10
Rab1A
Rab1B
Rab2
Rab3A
Rab3B
Rab3C
Rab3D
Rab4
Rab5A
Rab5B
Rab5C
Rab6
Rab7
Rab8
Rab9
Rab10
Rab11
Rab12
Rab13
Rab14
Rab15
Rab16
Rab17
Rab18
Ras1
Ras2
Rsr1
Rho1
Rho2
Rho3
Rho4
Cdc42
Sec4
Ypt1
Ypt2
Ypt3
Ypt4
Ypt5
Ypt6
Ypt7
Ypt8
Arf
Sar1
Ran
Arf1
Arf2
Arf3
Arf4
Arf5
Arf6
Sar1A
Sar1B
Ran
Arf1
Arf2
Arf3
Sar1
Gsp1
Gsp2
Mammal
Rab19
Rab20
Rab21
Rab22
Rab23
Rab24
Rab25
Rab26
Rab27
Yeast
protein involved in vesicle trafficking from the Golgi
complex to the plasma membrane (Salminen & Novick
1987). On the other hand, the Ypt1 gene was isolated
as a gene encoding a Ras-like G protein (Gallwitz et al.
1983). Ypt1 was also shown to be involved in vesicle
trafficking from ER to Golgi (Segev et al. 1988).
Thereafter, evidence has accumulated that the Rab
family members are involved in vesicle trafficking, not
Figure 1 Four principal mechanisms of intracellular vesicle
trafficking.
616 Genes to Cells (1996) 1, 615–632
only in yeast but also in mammal (Simons & Zerial
1993; Nuoffer & Balch 1994; Pfeffer 1994). There are
more than 30 members in the mammalian Rab family
and each member is located in each membrane
compartment and exerts its specific function as
shown in Fig. 2 and Table 2.
The precise mechanism of the Rab family members
in regulating vesicle trafficking has not been fully
understood, but one group of the Rab family members
regulate the budding process and another group
regulates the targeting and docking processes. In the
targeting and docking processes, the Rab family
members are assumed to regulate the assembly of the
SNARE components, which is also involved in the
targeting and docking processes of the vesicle with
the acceptor membrane. This docking process is
followed by the fusion process, which is mediated by
the general fusion machinery, named the NSF/SNAP
system (NSF ˆ N -ethylmaleimide-sensitive fusion
protein; SNAP ˆ soluble NSF attachment protein;
and SNARE ˆ SNAP receptor.) The NSF/SNAP
and SNARE systems have been reviewed previously
(Rothman 1994). However, it remains to be clarified
how the Rab family members regulate the SNARE
system.
Of the many Rab family members involved in the
targeting and docking processes, the mode of action of
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Rab3A and Ca2+-dependent exocytosis
Figure 2 Subcellular localization of each
Rab family member. Plain, localization of
each Rab family member; Italic, functioning site of each Rab family member.
Rab3A (a member of the Rab3 subfamily) has been
extensively investigated, and evidence has accumulated
that Rab3A is involved in Ca2+-dependent exocytosis,
particularly in neurotransmitter release. Moreover, the
regulators of Rab3A, named Rab GDP dissociation
inhibitor (Rab GDI), Rab3A GDP/GTP exchange
protein (Rab3A GEP), Rab3A GTPase activating
protein (Rab3A GAP), and a downstream target of
Rab3A named rabphilin3, have been isolated and
characterized. In this review article, we focus on
Rab3A and molecules which interact with it and
describe their functions and modes of action in
neurotransmitter release.
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Rab3A
Isolation of Rab3A
Rab3A is a member of the Rab3 subfamily consisting of
four members, Rab3A, Rab3B, Rab3C and Rab3D. All
of these members show calculated Mrs of about 25 000.
Rab3A was found by two different methods: in one, a
small G protein was purified from bovine brain and its
partial amino acid sequence was determined (Kikuchi
et al. 1988). On the basis of this information, its cDNA
was cloned from a bovine brain cDNA library and its
primary structure was determined (Matsui et al. 1988).
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Y Takai et al.
Table 2 Function of each Rab family
member
Protein
Vesicle trafficking
Rab1A
Rab1B
Rab8
Rab9
ER to Golgi
ER to cis Golgi
Cis Golgi to medial Golgi
ER to Golgi
Ca2+-dependent exocytosis
Early endosome-plasma membrane recycling
Plasma membrane to early endosome
Fusion of early endosome
Trans Golgi network to basolateral plasma membrane
Late endosome to trans Golgi network
Ypt1
Ypt7
Sec4
Sar1
ER to Golgi
Endocytosis
Post-Golgi to plasma membrane
ER to Golgi
Mammal
Rab2
Rab3A
Rab4A
Rab5A
Yeast
During this cloning, two other highly homologous
cDNAs were cloned and their primary structures were
determined. They were named Rab3B and Rab3C. In
the second method, Rab3A was isolated from a rat brain
cDNA library by the use of several oligonucleotide
mixtures corresponding to the conserved region of the
Ras subfamily members as a probe (Touchot et al. 1987).
Rab3D was later cloned as a Rab3-like molecule from
the differentiated 3T3-l adipocyte-specific subtractive
library (Baldini et al. 1992). The human Rab3A gene has
been shown to be located in chromosome 19, with a
regional localization on 19p13.2 (Rousseau-Merck et al.
1989).
The geranylgeranyl moiety is donated by geranylgeranylpyrophosphate by the action of geranylgeranyltransferase type II. The methyl moiety is
donated by S-adenosylmethionine by the action of
methyltransferase. These lipid modifications are essential for the interaction of Rab3A with the membrane
and its regulators as described below. It is not currently
known whether the post-translational modifications of
the Rab3 subfamily members turn over rapidly in
reponse to an agonist, but it is generally believed that
the geranylgeranylation of small G proteins is an
irreversible reaction, whereas the carboxylmethylation
is a reversible reaction in intact cells.
Structure of Rab3A
Tissue and subcellular distributions of Rab3A
As shown in Fig. 3, all the four Rab3 subfamily
members have consensus amino acid sequences for
GDP/GTP-binding and GTPase activities as have been
described for other G proteins. These sequences
compromise four regions: (1) (Gly-X-X-X-X-GlyLys), (2) (Asp-Thr-Ala-Gly), (3) (Asn-Lys-X-Asp), and
(4) (Glu-X-Ser-Ala-X) (X: any amino acid). Regions 1
and 2 are responsible for GTPase activity and regions 3
and 4 for GDP/GTP-binding activity. Moreover, all the
four Rab 3 subfamily members have a unique Cterminal structure of Cys-X-Cys, which undergoes
post-translational modifications with lipids: the two
cysteine residues are geranylgeranylated through
thioether linkage; the C-terminal cysteine residue is
further carboxylmethylated (Farnsworth et al. 1991).
Northern and Western blot analyses indicate that
Rab3A is detected in cells having a regulated secretion
pathway (including neuronal cells, exocrine cells and
endocrine cells) and not in cells having only a
constitutive secretion pathway (including hepatocytes
and lymphocytes) (Mizoguchi et al. 1989; Sano et al.
1989). In situ hybridization has revealed that the Rab3A
and Rab3B mRNAs are colocalized in most neurones in
all brain areas examined, but that, in each of these areas,
subsets of neurones preferentially express either the
Rab3A or Rab3B mRNA (Stettler et al. 1995). In
neuroendocrine PC12 cells, Rab3A is expressed, but
Rab3B is not (Weber et al. 1994). In the brain, Rab3A is
highly concentrated on synaptic vesicles in the presynaptic nerve terminal (Fischer von Mollard et al. 1990;
618 Genes to Cells (1996) 1, 615–632
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Rab3A and Ca2+-dependent exocytosis
Figure 3 Structures of Rab3 subfamily
members. 1 and 2, consensus sequences for
GTPase activity; 3 and 4, consensus
sequences for GDP/GTP-binding activity.
Lines in Rab3B, Rab3C and Rab3D
indicate the different amino acids from
Rab3A.
Mizoguchi et al. 1990). Immunoelectromicroscopic
analysis on the subcellular localization of Rab3A in
neuromuscular junctions indicates that Rab3A is
uniformly distributed over synaptic vesicles in the
nerve terminal of motor neurones (Mizoguchi et al.
1994). In adrenal medulla chromaffin cells, Rab3A is
present on synaptic-like microvesicles and secretory
granules (Darchen et al. 1990, 1995; Fischer von
Mollard et al. 1990).
Two interconvertible forms of Rab3A
Like other G proteins, Rab3A has two interconvertible
forms: GDP-bound and GTP-bound, as shown in Fig. 4.
The GDP-bound form is inactive and the GTP-bound
form is active and interacts with its downstream target.
Rab3A receives an upstream signal, is converted from the
GDP-bound form to the GTP-bound form and
transduces a signal to the downstream target. The GDPbound form is converted to the GTP-bound form by a
GDP/GTP exchange reaction, whereas the GTP-bound
form is converted to the GDP-bound form by a GTPase
reaction. The rate-limiting step of the GDP/GTP
exchange reaction is the dissociation of GDP from the
GDP-bound form (Shoji et al. 1989). Once GDP
dissociates from the GDP-bound form, the guanine
Figure 4 Modes of activation and action
of Rab3A.
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Y Takai et al.
nucleotide-free form is transiently produced. Since the
intracellular concentration of GTP is more than 100fold higher than that of GDP, GTP binds to the
guanine nucleotide-free form, resulting in the formation
of the GTP-bound form. Rab3A has intrinsic GTPase
activity, and GTP bound to Rab3A is hydrolysed
intramolecularly, resulting in the formation of the GDPbound form.
Recent kinetic analysis on the interaction of GDP and
GTP with the lipid-modified and unmodified forms of
Rab3A indicates that the lipid modifications of Rab3A
intramolecularly affect the interaction of GTP with
Rab3A (unpublished data). GDP dissociates from both
the lipid-modified form and the lipid-unmodified form
at similar rates. In contrast, GTP binds to the lipidmodified, guanine nucleotide-free form of Rab3A more
slowly than it binds to the lipid-unmodified, guanine
nucleotide-free form. Moreover, GTP rapidly dissociates from the lipid-modified form of Rab3A, whereas it
practically does not dissociate from the lipid-unmodified
form. This slow binding of GTP to the lipid-modified,
guanine nucleotide-free form of Rab3A is markedly
stimulated by Rab3A GEP, and this rapid dissociation of
GTP from the lipid-modified form of Rab3A is
markedly inhibited by dimyristoyl phosphatidylcholine
(DMPC).
It has been shown that GTP dissociates more rapidly
from the lipid-modified form of Arf1 than from the
lipid-unmodified form, and that this rapid dissociation is
inhibited by phospholipid (Franco et al. 1995). Arf1 is
myristoylated at its N-terminal region; three-dimensional structural analysis of the GDP/GTP-binding
region of Arf1 indicates that the N-terminal region is
located near its GDP/GTP-binding region (Amor et al.
1994). Therefore the lipid moiety may interact
intramolecularly with the GDP/GTP-binding region
and affect its conformation. The three-dimensional
structural analysis of Rab3A has not been carried out,
but it is possible that the lipid moieties of the C-terminal
region of Rab3A also interact with its GDP/GTPbinding region and affect its conformation. Rab3A GEP
and phospholipid may interact with the lipid moieties of
Rab3A and inhibit their effect on the GDP/GTPbinding region. It is unknown which modification,
geranylgeranylation or methylation, is involved in this
function.
Regulators of Rab3A
Interconversion of Rab3A between its two forms is
regulated as shown in Fig. 4. There are two types of
620 Genes to Cells (1996) 1, 615–632
regulators which regulate the GDP/GTP exchange
reaction of Rab3A: one inhibits the reaction and
is named Rab GDI, whereas the other stimulates it
and is named Rab3A GEP (GEP is also called GDP/
GTP exchange factor (GEF) or guanine nucleotide
releasing factor (GNRF)). There is one regulator that
stimulates the GTPase reaction of Rab3A, named
Rab3A GAP.
Rab GDI
Isolation and structure of Rab GDI
Rab GDI constitutes a family consisting of at least three
members: Rab GDI, GDI and GDI . All of these
members show calculated Mrs of about 50 000 (Matsui
et al. 1990; Nishimura et al. 1994; Shisheva et al. 1994).
Rab GDI was first purified from the bovine brain and its
partial amino acid sequence was subsequently determined (Sasaki et al. 1990). On the basis of this
information, its cDNA was cloned from a bovine brain
cDNA library and its primary structure was determined
(Matsui et al. 1990). Rab GDI and GDI were isolated
from the rat brain and mouse skeletal muscle cDNA
libraries, respectively, by the use of Rab GDI as a probe
with low stringency (Nishimura et al. 1994; Shisheva et al.
1994). Rab GDI is specifically expressed in the brain,
whereas Rab GDI is expressed in all tissues examined
(Nishimura et al. 1994). The tissue distribution of Rab
GDI has not been examined. The human Rab GDI
gene has been shown to be located in chromosome X,
with a regional localization on Xq28 (Sedlacek et al.
1994). Rab GDI is found in yeast and Drosophila, as
well as in mammal, and is named GDI1 and dGDI,
respectively, (Zahner & Cheney 1993; Garrett et al.
1994). GDI1 and dGDI show 50% and 68% amino acid
sequence identities with bovine Rab GDI. Recent
Southern blot analysis has shown that there are at least five
genes of Rab GDI in both the rat and mouse genomes
(Janoueix-Lerosey et al. 1995). Rab GDI may constitute a
large family.
The Rab GDI family has a homology to Rab-escort
protein, which delivers the newly synthesized Rab
family members to the catalytic subunit of geranylgeranyltransferase type II (Seabra et al. 1992, 1993; Andres
et al. 1993; Cremers et al. 1994). Rab-escort protein is
referred to as the choroideremia gene product, which,
when defective, contributes to a human degenerative
retinal disease (Cremers et al. 1992; Seabra et al. 1992,
1993; Andres et al. 1993; von Bokhoven et al. 1994).
Very recently, the three-dimensional structure of Rab
GDI has been determined (Schalk et al. 1996). The
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Rab3A and Ca2+-dependent exocytosis
structure of Rab GDI is remarkably similar to FADcontaining monooxygenases and oxidases, although its
physiological significance is not clear. Two of the
sequence regions conserved with the Rab-escort protein
form a compact structure at the apex of Rab GDI, and
may contribute to its unique biochemical properties, as
described below.
Unique biochemical properties of Rab GDI
Purified Rab GDI is a soluble protein and shows
unique biochemical properties: it interacts with the lipidmodified, GDP-bound form of Rab3A to form a stable
ternary complex, but not with any other forms such as
the lipid-modified, GTP-bound form, the lipid-unmodified, GDP-bound form or the lipid-unmodified, GTPbound form (Araki et al. 1990, 1991). Moreover,
Rab GDI only inhibits the dissociation of GDP
from the lipid-modified form of Rab3A. The lipidmodified form of Rab3A, irrespective of the GDPbound form and the GTP-bound form, but not the
lipid-unmodified form, are associated with any membrane, such as erythrocyte ghosts and synaptic vesicles, in
a cell-free system. Rab GDI inhibits this association of
only the GDP-bound form by forming the ternary
complex. Moreover, Rab GDI induces the dissociation
of only the lipid-modified, GDP-bound form of
Rab3A, which is prebound to the membranes, by
formation of the ternary complex. These biochemical
properties confer two activities on Rab GDI: one is to
regulate the GDP/GTP exchange reaction of Rab3A;
the other is to regulate the reversible binding of Rab3A
to membranes. On the other hand, Rab GDI has been
shown to be active, not only on Rab3A, but also on all
the Rab family members thus far examined, including
yeast Sec4 and Ypt1 (Sasaki et al. 1991; Garrett et al.
1993; Soldati et al. 1993; Ullrich et al. 1993; Beranger
et al. 1994). The biochemical properties of Rab GDI,
including the substrate specificity and requirement of the
lipid modifications of their substrates, are similar
(Nishimura et al. 1994; Araki et al. 1995). The biochemical properties of Rab GDI have not been studied,
but Rab GDI has been shown to exhibit a unique
intracellular distribution. Although Rab GDI is a
soluble protein, Rab GDI is located in the perinuclear
region of differentiated 3T3-L1 adipocytes (Shisheva et al.
1994, 1995). It is yet to be clarified whether there are
functional differences among the isoforms of Rab GDI.
Mode of action of Rab GDI
An attractive model for the mode of action of the Rab
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Figure 5 (A) A model for the mode of action of Rab family
members in targeting and docking processes in vesicle trafficking.
(B) A model for the mode of action of Rab GDI in Rab memberregulated vesicle trafficking.
family members in the targeting and docking processes
in vesicle trafficking was proposed (Bourne 1988). This
model was based on the mode of action of elongation
factor Tu in protein synthesis. As shown in Fig. 5A,
the GDP-bound form of each Rab family member
is located in the cytosol. When it is converted to the
GTP-bound form, it binds to a vesicle which is then
transported to the acceptor membrane. According to
this model, GTP hydrolysis occurs before the fusion
of the vesicle with the membrane. After the hydrolysis, the GDP-bound form is translocated to the
cytosol.
In this model, two types of cycling of each Rab family
member between the GDP-bound form and the GTPbound form, and between the cytosol and the
membrane, are essential for their action. Because Rab
Genes to Cells (1996) 1, 615–632 621
Y Takai et al.
GDI is a soluble protein and has the potential to regulate
these two types of cycling, we proposed the mode of
action of Rab GDI in the Rab family member-regulated
vesicle trafficking as shown in Fig. 5B: the GDP-bound
form of each Rab family member is complexed with
Rab GDI and remains in the cytosol. After it is released
from Rab GDI and is converted to the GTP-bound
form, or after it is converted to the GTP-bound form
and is released from Rab GDI, the GTP-bound form is
associated with the vesicle, which is consequently
transported to the acceptor membrane. Before the
fusion of the vesicle with the membrane, the GTPbound form is converted to the GDP-bound form.
Once the GDP-bound form is produced on the
membrane, it is complexed with Rab GDI and is
translocated from the membrane to the cytosol. The
Rab family members are also assumed to cycle between
the GDP-bound and the GTP-bound form and
between the cytosol and the membrane in the budding
process of vesicle trafficking. The model shown in Fig.
5B is also applicable to this budding process.
Several lines of evidence supporting this model have
accumulated: (i) many Rab family members, including
Rab1, Rab3A, Rab6 and Rab9, have been shown to be
complexed with Rab GDI in the cytosol of various
tissues and cells (Regazzi et al. 1992; Soldati et al. 1993;
Peter et al. 1994; Yang et al. 1994); (ii) genetic analysis of
yeast has shown that Rab GDI indeed regulates the Sec4
function (Garrett et al. 1994); (iii) it has been proved by
use of cell-free assay systems for vesicle trafficking
between ER and Golgi complex and inter-cisternal
vesicle trafficking in Golgi complex, that Rab GDI
indeed functions in these systems (Elazar et al. 1994;
Peter et al. 1994). Recent studies using Rab5 and Rab9
have shown that Rab GDI has an additional function to
transport these small G proteins to the membrane,
where they are converted to the GTP-bound form
(Dirac-Svejstrup et al. 1994; Soldati et al. 1994; Ullrich
et al. 1994). The Rab family members may cycle by the
action of Rab GDI in the cytosol in the immediate
vicinity of the membrane. It has also been shown by use
of Rab5 that Rab GDI serves as a cytosolic acceptor for
the newly synthesized, lipid-modified Rab family
members (Sanford et al. 1995). The Rab family
members may be transferred from Rab-escort protein
to Rab GDI after the lipid modifications.
Rab3A GEP
Conversion of the GDP-bound form of Rab3A to the
GTP-bound form is regulated by GEP. Thus far two
GEPs for Rab3A have been reported: one is a GEP,
622 Genes to Cells (1996) 1, 615–632
named MSS4 (Burton et al. 1993), which was isolated as
a mammalian counterpart of yeast DSS4 (a GEP for
yeast Sec4 and Ypt1 (Moya et al. 1993)); and the other is
a GEP, named Rab3A GRF (Burstein & Macara 1992),
which was partially purified from rat brain. MSS4 shows
a calculated Mr of about 14 000 (Burton et al. 1993).
Recombinant MSS4 is not only active on Rab3A but
also on other Rab family members, including Rab1,
Rab8, Rab10, Sec4 and Ypt1 (Burton et al. 1994).
Recombinant MSS4 is equally active on both the lipidmodified and unmodified forms of Rab3A (Miyazaki
et al. 1994). The action of MSS4 is inhibited by Rab
GDI, with the lipid-modified form of Rab3A as a
substrate (Miyazaki et al. 1994). Rab3A GRF is also
sensitive to the action of Rab GDI, but it prefers the
lipid-modified form of Rab3A to the lipid-unmodified
form (Burstein et al. 1993; Miyazaki et al. 1994). The
substrate specificity of Rab3A GRF has not been studied
intensively.
A GEP specific for Rab3A has recently been
highly purified from rat brain (unpublished data).
There are two isoforms, named Rab3A GEPI and
GEPII. Rab3A GEPI and GEPII demonstrate similar
physical and kinetic properties. Both show Mrs of about
200 000 and 270 000 as estimated by sodium dodecylsulphate–polyacrylamide gel electrophoresis (SDSPAGE) and gel filtration, respectively. Rab3A GEPI
and GEPII are active on the Rab3 subfamily members
and inactive on other Rab family members so far
examined, including Rab2, Rab5, Rab10 and Rab11.
They are active on the lipid-modified form of
Rab3A and inactive on the lipid-unmodified form.
Their action is inhibited by Rab GDI. It remains to be
clarified whether Rab3A GEPs are identical with
Rab3A GRF. It is also not known which GEP functions
in vivo for activating Rab3A, but Rab3A GEP may
be the most probable candidate because it is specific for
the Rab3 subfamily.
The precise mechanism for the mode of action of
Rab3A GEP in the GDP/GTP exchange reaction of
Rab3A is not known, but on the assumption that the
mode of action of Rab3A GEP is principally similar to
that of well-characterized Ras GEPs, such as Sos and
Cdc25, the following mechanism is conceivable: Rab3A
GEP forms a complex with the GDP-bound form of
Rab3A and stimulates the dissociation of GDP from
Rab3A to produce a complex of Rab3A GEP with the
guanine nucleotide-free form. For the interaction of
Rab3A GEP with the GDP-bound form of Rab3A
and the guanine nucleotide-free form, the lipid
moieties of Rab3A may be necessary. The Rab3A
GEP in this complex is then replaced by GTP to
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Rab3A and Ca2+-dependent exocytosis
produce the GTP-bound form. In this way, Rab3A
GEP catalytically stimulates the GDP/GTP exchange
reaction of Rab3A. The binding of GTP to the guanine
nucleotide-free, lipid-modified form of Rab3A is very
slow—this binding is facilitated by Rab3A GEP,
presumably by masking the lipid-moieties (unpublished
data).
Rab GDI displacement factor
The GDP-bound form of Rab3A complexed with Rab
GDI is not converted to the GTP-bound form by the
action of any of the Rab3A GEPs described above,
suggesting that another factor is also necessary for this
conversion. It has been shown that the GDP-bound
form of Rab9 complexed with Rab GDI is converted to
the GTP-bound form when the complex is incubated
with the endosome membrane (Soldati et al. 1994). On
the basis of this observation, it has been proposed that
a membrane factor is necessary for the dissociation of
the GDP-bound form of Rab9 from Rab GDI—this
factor is named GDI displacement factor (GDF) (Soldati
et al. 1994). A similar result has also been obtained
for Rab5 (Ullrich et al. 1994). GDF may also function
in recruiting Rab GDI to the membrane. GDF has
not been identified, and it is not known whether GDF
is a specific regulator of each Rab family member or
a general regulator of all the Rab family members
like Rab GDI. If the former is the case, a GDF specific
for Rab3A may be present.
Rab3A GAP
Conversion from the GTP-bound form of Rab3A to
the GDP-bound form is regulated by GAP. Rab3A GAP
was first purified partially from the rat brain (Burstein
et al. 1991), but its properties have not been studied
intensively. A GAP specific for Rab3A has recently been
highly purified and characterized from the rat brain
(unpublished data). The purified sample of Rab3A GAP
consists of two subunits with Mrs of about 130 000 and
150 000, as estimated by SDS-PAGE. The protein shows
a Mr of about 300 000 both by gel filtration and by
sucrose density gradient ultracentrifugation, suggesting
that Rab3A GAP is a heterodimeric protein. The
small Mr size protein appears to be the catalytic subunit.
Rab3A GAP is active on the Rab3 subfamily members,
but inactive on other Rab family members so far
examined, including at least Rab2, Rab5 and Rab11. It
is active on the lipid-modified form of Rab3A but
is inactive on the lipid-unmodified form. It is not
known when and how the GTP-bound form of
# Blackwell Science Limited
Rab3A is converted to the GDP-bound form by the
action of Rab3A GAP, but this point will be discussed
below.
Downstream targets of Rab3A
Rabphilin3
Isolation of rabphilin3
Once the GTP-bound form of Rab3A is produced, it
appears to interact specifically with synaptic vesicles,
because Rab3A is specifically localized on synaptic
vesicles. It is assumed that this specific localization of
the GTP-bound form of Rab3A is due to the presence of a specific protein on synaptic vesicles, which
interacts with the GTP-bound form of Rab3A. On
this assumption, this downstream target of Rab3A was
detected by the use of a cross-linking technique
(Shirataki et al. 1992). One protein, which was
specifically cross-linked with the GTP-bound form of
radioiodinated Rab3A, was detected in the bovine
brain crude membrane fraction. This cross-linking was
competitively inhibited by the GTP-bound form
of nonradioiodinated Rab3A, but not by the GDPbound form. By the use of this assay method, this
protein was purified and its partial amino acid
sequence was determined. On the basis of this
information, its cDNA was cloned from a bovine
brain cDNA library and its primary structure was
determined (Shirataki et al. 1993). This protein was
named rabphilin-3A. Recombinant rabphilin-3A
indeed prefers the GTP-bound form of Rab3A to the
GDP-bound form. It also interacts with the GTPbound form of Rab3B and Rab3C, but does not
interact with other Rab family members, including at
least Rab1B, Rab5 and Rab11 (Li et al. 1994; Weber
et al. 1996; and our unpublished data). It may therefore
be better to change the name of rabphilin-3A to
rabphilin3. Rabphilin3 has also been found in Caenorhabditis elegans (Brose et al. 1995).
Structure of rabphilin3
Rabphilin3 has a calculated Mr of about 78 000.
Structural analysis indicates that rabphilin3 has no
transmembrane segment but has two C2-like domains
(C2A domain and C2B domain) at its C-terminal
region, as shown in Fig. 6 (Shirataki et al. 1993). The C2
domain was originally found in protein kinase C, which
is activated by Ca2+ and phospholipid, particularly
phosphatidylserine (Nishizuka 1988). Protein kinase C
has one C2 domain, but synaptotagmin has been
shown to have two C2-like domains (C2A domain
Genes to Cells (1996) 1, 615–632 623
Y Takai et al.
Figure 6 Structures of rabphilin3, protein
kinase C and synaptotagmin.
and C2B domain) and to interact with Ca2+ and
phospholipid, particularly phosphatidylserine (Perin
et al. 1990). This protein constitutes a family consisting
of at least eight members (Li et al. 1995). The originally
isolated member, synaptotagmin I, is specifically
localized on synaptic vesicles through its one transmembrane segment and has been suggested to serve as a Ca2+
sensor for neurotransmitter release (Perin et al. 1990;
Brose et al. 1992; Geppert et al. 1994b). Recombinant
rabphilin3 actually interacts with Ca2+ and phospholipid,
particularly phosphatidylserine, at the C2-like domains,
whereas it interacts with Rab3A at its N-terminal region
(Yamaguchi et al. 1993). Thus, rabphilin3 has at least two
functionally different domains, the N-terminal Rab3Abinding domain and the C-terminal Ca2+- and
phospholipid-binding C2-like domain. We named
these domains the Rab3 domain and the C2 domain,
respectively. Recent analysis indicates that Rab3A
interacts with amino acid residues 40–170 of rabphilin3
(Stahl et al. 1996). This region has two repeats of a CysX2-Cys (X: any amino acid) structure which are also
found in the region of Raf protein kinase which interacts
with Ras and Rap1.
In addition to these two domains of rabphilin3, the
middle region shown in Fig. 6 is rich in prolines and
serine/threonine phosphorylation sites and we have
called it the M domain. Indeed, rabphilin3 is phosphorylated by protein kinase A and calmodulindependent protein kinase II (CaMKII) (Kato et al.
624 Genes to Cells (1996) 1, 615–632
1994; Numata et al. 1994; Fykse et al. 1995) in a cell-free
system. We have shown that Ser34, Thr205, Thr209 and
Thr537 are phosphorylated by CaMKII, whereas
another group has shown that Ser234 and Ser274 are
phosphorylated. The reason for these different results
is unknown, but may be due to different experimental conditions. The function of the phosphorylation of rabphilin3 remains to be clarified, but
four of the six phosphorylation sites are located at the
M domain and this domain may also have its own
specific function.
Tissue and subcellular distributions of rabphilin3
Northern and Western blot analyses indicate that
rabphilin3 is expressed in neuronal cells, but not in
other cells, including exocrine cells and endocrine
cells where Rab3A is expressed (Shirataki et al. 1993). In
the cells where Rab3A is expressed but rabphilin3 is
not, a rabphilin3 isoform may be present. In the brain,
rabphilin3 is highly concentrated on synaptic vesicles
in the presynaptic nerve terminal (Mizoguchi et al.
1994). Immunoelectromicroscopic analysis of the
subcellular distribution of rabphilin3 in neuromuscular junctions indicates that it is uniformly
distributed to most synaptic vesicles in the nerve
terminal of motor neurones and that its distribution
is similar to that of Rab3A (Mizoguchi et al. 1994).
We have shown that rabphilin3 interacts with isolated
synaptic vesicles in dose-dependent and saturable
# Blackwell Science Limited
Rab3A and Ca2+-dependent exocytosis
manners (Shirataki et al. 1994). This interaction is
observed when the synaptic vesicles are previously
deprived of Rab3A by the use of Rab GDI or associated
with large amounts of exogenous Rab3A, but is
abolished by prior digestion of the vesicles with trypsin.
These results suggest that rabphilin3 is associated with
the vesicles through its anchoring protein, irrespective of
the presence or absence of Rab3A. This result is
consistent with the structural property of rabphilin3 that
it has no transmembrane segment. Therefore, it is
likely that the specific localization of rabphilin3 is
determined by the specific localization of its anchoring
protein. In contrast to our results, however, another
group has suggested that rabphilin3 is associated with
synaptic vesicles through Rab3A, on the basis of
the observations that, in Rab3A-deficient mice,
rabphilin3 was decreased in synapses (Li et al. 1994)
and that Rab GDI removed both Rab3A and rabphilin3
from isolated synaptic vesicles (Stahl et al. 1996).
However, these studies have not shown directly that
rabphilin3 interacts with the vesicles in a Rab3Adependent manner, whereas we have directly shown
that rabphilin3 interacts with the vesicles in a Rab3Aindependent manner.
Evidence for the involvement of the Rab3
subfamily members in vesicle trafficking
There are several lines of evidence supporting the
involvement of the Rab3 subfamily members in vesicle
trafficking. In isolated nerve terminals, the stimulation
of neurotransmitter release results in dissociation of
Rab3A and Rab3C from synaptic vesicles (Fischer von
Mollard et al. 1991, 1994), although inconsistent results
have also been reported (Bielinski et al. 1993). When
synthetic peptides corresponding to the putative effector
domain of the Rab3 subfamily members are introduced
into secretory cells, such as exocrine pancreas (Padfield
et al. 1992; Edwardson et al. 1993; Li et al. 1993), mast
cells (Oberhauser et al. 1992), chromaffin cells (Senyshyn et al. 1992) and cultured neurones (Richmond &
Haydon 1993), they stimulate secretion, although
opposite results have also been reported (Davidson et al.
1993), and several recent studies moreover suggested
that the effects of these peptides could be attributed to
another mechanism unrelated to the Rab3 subfamily
members (Law et al. 1993; Piiper et al. 1993). The
transient overexpression of a dominant active mutant of
Rab3A has been shown to inhibit Ca2+-dependent
secretion from bovine chromaffin cells and PC12 cells
(Holz et al. 1994; Johannes et al. 1994). A similar
# Blackwell Science Limited
conclusion has been also reached when a dominant
active mutant of Rab3A is microinjected into bovine
chromaffin cells (Johannes et al. 1994). In contrast to the
inhibitory effect of Rab3A, Rab3B has been shown to
stimulate secretion. In antisense RNA experiments
performed on anterior pituitary cells, reductions in
Rab3B mRNA inhibits Ca2+-dependent secretion from
these cells (Lledo et al. 1993). When a dominant active
mutant of Rab3B is stably expressed in PC12 cells,
where endogenous Rab3B is absent, the Ca2+-dependent norepinephrine secretion is markedly stimulated
(Weber et al. 1996). The reason for these apparently
opposite effects of Rab3A and Rab3B on the Ca2+dependent exocytosis is currently unknown, but these
results, together with those of tissue and subcellular
distributions of the Rab3 subfamily members, and by
analogy with the functions of Sec4 and Ypt1, suggest
that the Rab3 subfamily members are involved in Ca2+dependent exocytosis.
Evidence for the involvement of
rabphilin3 in Ca2+-dependent
neurotransmitter release
Because rabphilin3 interacts with the GTP-bound form
of Rab3A, is specifically localized on synaptic vesicles
and interacts with Ca2+, it is likely that rabphilin3, as well
as synaptotagmin, plays a role in Ca2+-dependent
neurotransmitter release. This assumption has recently
been proven by the use of a human growth hormone
(GH) co-expression assay (Chung et al. 1995; Komuro
et al. 1996). In this system, GH is overexpressed in
bovine adrenal chromaffin cells or PC12 cells. Exogenous GH is stored in dense core vesicles and released in
response to an agonist in an extracellular Ca2+dependent manner. When the N-terminal fragment of
rabphilin3 shown in Fig. 6 is co-overexpressed with
human GH in bovine adrenal chromaffin cells, the
Ca2+-dependent, agonist-induced GH release is markedly inhibited. In contrast, overexpression of the Cterminal fragment, which contains both a part of the M
domain and the C2 domain, has no effect on GH
release. Similar results have also been obtained by the use
of PC12 cells (Komuro et al. 1996). However, in the
PC12 cell experiment, overexpression of the C2
fragment inhibits the Ca2+-dependent, high K+induced GH release. The exact reason for the different
effects of these two fragments is not known, but it may
be due to the inhibitory effect of the M domain on the
C2 domain. It has furthermore been shown that
overexpression of the C2B fragment, but not the
Genes to Cells (1996) 1, 615–632 625
Y Takai et al.
Figure 7 A model for the modes of action of the Rab3A system, the synaptotagmin system and the NSF/SNAP and SNARE systems in
neurotransmitter release.
C2A fragment, inhibits secretion in the PC12 cell
experiment.
Evidence for the involvement of rabphilin3 in Ca2+dependent exocytosis has also been obtained by the use
of a squid giant axon system, in which the presynaptic
nerve terminal is large enough to be microinjected with
the protein samples to be tested (unpublished data). The
N-terminal fragment or the C2 fragment is microinjected into the nerve terminal whilst electrically
stimulating this terminal. Postsynaptic electrical
responses are recorded simultaneously to monitor
neurotransmitter release. Microinjection of the Nterminal fragment or the C2 fragment inhibits Ca2+dependent neurotransmitter release. Electron microscopy has revealed that injections cause a reduction in
the number of synaptic vesicles within 0.5 m of the
plasma membrane, and a relative accumulation of predocked vesicles 50–100 nm away from the plasma
membrane. In contrast, while microinjection of the
peptides from the C2-like domains of synaptotagmin
also inhibits Ca2+-dependent neurotransmitter release,
this perturbation causes an accumulation of docked
vesicles within 50 nm of the plasma membrane
(Bommert et al. 1993). These ultrastructural data
suggest that rabphilin3 is involved in the pre-docking
626 Genes to Cells (1996) 1, 615–632
process or the docking process, whereas synaptotagmin
is involved in the fusion process that follows the
docking process. These two lines of evidence indicate
that the Rab3A-rabphilin3 system indeed functions in
Ca2+- dependent exocytosis in intact cells.
Possible function of rabphilin3 as a linker
for the Rab3 system, the NSF/SNAP and
SNARE systems and the Ca2+ system
The exact mode of action of synaptotagmin in Ca2+dependent neurotransmitter release has not been
established, but one fascinating function proposed is
that synaptotagmin serves as a Ca2+-controlled negative
regulator, as shown in Fig. 7. Namely, the docking and
fusion of synaptic vesicles with the presynaptic plasma
membranes are mediated by the NSF/SNAP and
SNARE systems. In the SNARE system, the component located on the vesicles is vesicle-associated
membrane protein (VAMP) and the components
located on the plasma membrane are syntaxin and the
25 kDa synaptosomal-associated protein (SNAP-25).
Before Ca2+ influx, synaptotagmin interacts with
syntaxin and inhibits the operation of the NSF/SNAP
# Blackwell Science Limited
Rab3A and Ca2+-dependent exocytosis
and SNARE systems. When Ca2+ influxes into the
cytoplasm through the Ca2+ channel, it binds to
synaptotagmin, causing the release of this inhibitory
action to operate the NSF/SNAP and SNARE systems.
A recent biochemical observation, however, shows that
the C2A domain of synaptotagmin interacts with
syntaxin in a Ca2+-dependent manner (Chapman et al.
1995; Li et al. 1995). It is therefore likely that
synaptotagmin interacts with syntaxin after Ca2+
influx and then operates the NSF/SNAP and SNARE
systems. In this case, synaptotagmin serves as a positive
regulator for the stimulation of Ca2+-dependent
exocytosis.
It is also not known how rabphilin3 regulates synaptic
vesicle trafficking, but evidence is accumulating that the
Rab system is an upstream regulator of the NSF/SNAP
and SNARE systems: (i) genetic analysis using the
budding yeast has shown that Ypt1 directly or indirectly
induces the assembly of the SNARE components (Lian
et al. 1994; Sogaard et al. 1994); (ii) inhibition of the
rabphilin3 function by microinjecting the N-terminal
fragment or the C2 fragment into squid giant axons
causes an accumulation of pre-docked vesicles 50–100
nm away from the plasma membrane as described above;
and (iii) a dominant active mutant of Rab3A inhibits
Ca2+-dependent exocytosis from chromaffin cells and
PC12 cells (Holz et al. 1994; Johannes et al. 1994). These
results suggest that the Rab3A–rabphilin3 system also
regulates the NSF/SNAP and SNARE systems in
neurotransmitter release. After the GTP-bound form
of Rab3A is converted to the GDP-bound form by the
action of Rab3A GAP, the Rab3A–rabphilin3 complex
may dissociate and the fusion is then induced by the
action of the NSF/SNAP and SNARE systems. This
model for the mode of action of Rab3A seems to be
consistent with the phenotype of Rab3A-deficient mice
(Geppert et al. 1994a). These mice show large decreases
in synaptic transmission only with repetitive stimulation,
for which sequential synaptic vesicle recruitment to the
presynaptic membrane is essential.
In addition to this function of rabphilin3, it may also
serve as a Ca2+ sensor, because rabphilin3 has two C2like domains, as does synaptotagmin. Our working
model for the mode of action of the Rab3A–rabphilin3
system in neurotransmitter release is shown in Fig. 7.
There is an acceptor protein directly or indirectly
recognized by rabphilin3, complexed with the GTPbound form of Rab3A on the presynaptic plasma
membrane. When rabphilin3 is complexed with the
GTP-bound form of Rab3A, it interacts with this
acceptor protein and thereby causes the docking of the
vesicles with the presynaptic plasma membrane. Thus,
# Blackwell Science Limited
rabphilin3 may have at least three functional domains:
the domains interacting with (i) Rab3A, (ii) an anchoring protein on synaptic vesicles and (iii) an acceptor
protein on the plasma membrane. This assumption is
consistent with the structural properties of rabphilin3—
that it has at least three domains—and also with the
results obtained by the GH overexpression experiment
and the squid giant axon experiment described
above—that both the N-terminal fragment and the
C2 fragment inhibit Ca2+-dependent exocytosis. The
Rab3A-dependent interaction of rabphilin3 with the
acceptor protein may be followed by the assembly of
synaptotagmin and SNARE components. Binding of
influxed Ca2+ to rabphilin3, as well as to synaptotagmin,
may cause either dissociation from or tighter interactions
with their respective interacting proteins and thereby
lead to the operation of the NSF/SNAP and SNARE
systems. The functional relationships between rabphilin3 and synaptotagmin and between rabphilin3 and the
NSF/SNAP and SNARE systems is unknown at
present, but rabphilin3 appears to be an important
linker for the Rab3 system, the NSF/SNAP and
SNARE systems and the Ca2+ systems.
Rabin3
A protein different from rabphilin3, which specifically
interacts with the GTP-bound form of Rab3A and
Rab3D but not with Rab3C, was isolated by the yeast
two-hybrid method and named Rabin3 (Brondyk
et al. 1995). It has a calculated Mr of about 50 000.
Rabin3 has a domain with sequence similarity to Sec2
which is essential for constitutive secretion in budding
yeast cells and interacts with Sec4 (Nair et al. 1990).
Rabin3 is ubiquitously expressed but particularly
abundant in testes. The function of rabin3 remains to
be clarified.
A possible mechanism for specific
localization of Rab3A on synaptic
vesicles
Immunoelectromicroscopic analysis indicates that both
Rab3A and rabphilin3 are uniformly distri-buted in
most synaptic vesicles at least in the nerve terminal of
motor neurones as described above. These results
suggest that rabphilin3 associated with synaptic vesicles
is always saturated with the GTP-bound form of
Rab3A. When rabphilin3 becomes free of the GTPbound form of Rab3A—after its conversion to the
GDP-bound form by the action of Rab3A GAP—it
Genes to Cells (1996) 1, 615–632 627
Y Takai et al.
may immediately interact with the GTP-bound form of
Rab3A which is newly converted from the GDP-bound
form complexed with Rab GDI. Rab3A GEP stimulates
the dissociation of GDP from Rab3A free from Rab
GDI, but not from Rab3A complexed with Rab GDI.
Even in the presence of rabphilin3, Rab3A GEP does
not act on Rab3A complexed with Rab GDI (our
unpublished data). It is likely that another factor, such as
Rab3 GDF, may be involved in the activation of Rab3A
complexed with Rab GDI.
The specific localization of Rab3A could be
determined by protein–protein interactions and not
only by lipid (from Rab3A)–lipid (from synaptic vesicle
membrane) interactions. Rabphilin3 specifically interacts with the GTP-bound form of the Rab3 subfamily
members and is specifically localized on synaptic
vesicles, it is likely that rabphilin3 determines the
specific localization of Rab3A. Rabphilin3 has been
shown to inhibit Rab3A GAP activity (Kishida et al.
1993), and the interaction of the lipid moieties of
Rab3A with phospholipid inhibits the dissociation of
GTP from Rab3A, as described above. Therefore, the
interactions of Rab3A with both rabphilin3 and vesicle
phospholipid may keep Rab3A in the GTP-bound
form until Rab3A accomplishes its function. It is also
possible that Rab3A GDF determines the specific
localization of Rab3A, if Rab3A GDF is specific for
the Rab3 subfamily members and is localized specifically
on synaptic vesicles.
Important functions of lipid-modifications
of Rab3A
As described above, the lipid modifications of Rab3A
are of crucial importance for its interaction with
regulators, including Rab GDI, Rab3A GEP and
Rab3A GAP. Moreover, they are essential for the
action of each regulator. Of these modifications, the
geranylgeranylation, but not carboxylmethylation, is
essential for the action of Rab GDI (Musha et al. 1992).
It is not known which modification is essential for the
actions of Rab3A GEP and Rab3A GAP. It is also not
known how these lipid modifications are important for
the actions of these regulators. Although the threedimensional structural analysis of Rab GDI has revealed
the structure responsible for its interaction with Rab3A,
as described above, it has not been determined which
structure of Rab GDI distinguishes the lipid-modified
form of Rab3A from the lipid-unmodified form. It is
possible that the lipid moieties interact directly with the
regulators or intramolecularly interact with Rab3A
628 Genes to Cells (1996) 1, 615–632
molecule itself so that it causes a conformational change
to interact with the regulators. Moreover, the lipid
moieties have been shown to intramolecularly affect the
interaction of GTP with Rab3A. Three-dimensional
structural analysis by crystallization of the lipid-modified
form of Rab3A and the regulators and their cocrystallizations are important for our understanding of
the function of the lipid moieties.
Conclusions and perspectives
Evidence is accumulating that Rab3A and its interacting
proteins are involved in Ca2+-dependent exocytosis,
particularly in neurotransmitter release. The Rab3A
system appears to play an important role as a linker for
the NSF/SNAP and SNARE systems and the Ca2+
system. On the basis of the evidence thus far available,
we propose a tentative model for the modes of action of
Rab3A and its interacting proteins in neurotransmitter
release, as shown in Fig. 7. In this model however, there
are still several issues to be clarified: (i) how is the GDPbound form of Rab3A staying in the cytosol,
complexed with Rab GDI, converted to the GTPbound form and transferred to rabphilin3 on synaptic
vesicles?; (ii) what is the anchoring protein of rabphilin3
on synaptic vesicles?; (iii) how does rabphilin3 complexed with the GTP-bound form of Rab3A cause the
targeting and/or docking of the vesicles to the
presynaptic plasma membrane?; (iv) what is the acceptor
protein of rabphilin3 on the presynaptic plasma
membrane?; (v) how does Ca2+ control the rabphilin3
and synaptotagmin functions?; (vi) when and how is the
GTP-bound form of Rab3A complexed with rabphilin3, converted to the GDP-bound form, and transferred to Rab GDI? The clarifications of these issues will
help our understanding of general mechanisms of the
Rab family member-regulated vesicle trafficking and
also of the mode of action of Ca2+ in Ca2+-dependent
exocytosis.
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