From the
Rabphilin3a had been identified in brain as a Rab3a-binding
protein and may serve as an effector for Rab3a function. We have cloned
a splice variant of brain-Rabphilin3a from a bovine adrenal chromaffin
cell cDNA library and investigated the function of the protein in
regulated exocytosis in bovine chromaffin cells. The predicted amino
acid sequence of chromaffin cell (c-) Rabphilin3a was identical with
that of brain (b-) Rabphilin3a except for a 6-amino-acid insert VFSLSA
in the amino-terminal half of the protein. An antibody directed against
a carboxyl-terminal peptide recognized an 85-kDa protein in COS7 cells
transfected with the cDNA in a mammalian expression vector. A band of
similar mobility was enriched in a fraction of highly purified
chromaffin granule membranes, consistent with the Rabphilin3a being
associated with chromaffin granule membranes. Overexpression of either
chromaffin cell or brain Rabphilin3a by transfection with the
corresponding cDNAs in mammalian expression vectors enhanced
DMPP-induced secretion of co-expressed human growth hormone (GH)
approximately 30%. Chromaffin cells transfected with a plasmid with the
entire coding sequence of c-Rabphilin3a inserted in the antisense
orientation inhibited secretion of co-expressed GH by approximately
30%. Rabphilin3a mutants lacking one or both of the carboxyl-terminal
C2 domains strongly inhibited DMPP-stimulated exocytosis. The single C2
domain deletion also strongly inhibited Ca
The convergence of genetic studies in yeast and biochemical
studies in mammalian cells has revealed two sets of proteins implicated
in vesicular trafficking including regulated exocytosis (reviewed in
Refs. 1-4). One set involves proteins of the SNARE
The other set of proteins consists of
members of the Rab class of low molecular weight GTPases. Distinct Rab
proteins are necessary for vesicular trafficking in the constitutive
secretory (7, 8, 9) and endocytosis
pathways(10, 11, 12, 13, 14) .
Although Rab family members are required for vesicular trafficking,
they are not core components of the SNARE complex. In mammalian cells
there is growing evidence that members of the Rab3 subclass of proteins
modulate regulated exocytosis(15, 16, 17) . We
and others have demonstrated that Rab3a acts as an inhibitory modulator
of secretion in bovine adrenal chromaffin (16, 17) and
PC12 (17) cells. We had found that overexpression of Rab3a inhibited
the earliest rates of Ca
Rabphilin3a was
identified in brain as a possible effector protein for Rab3a. It binds
specifically to the GTP-bound form of Rab3a(20, 21) and
also binds to Rab3c(22) . Cloning of bovine brain Rabphilin3a
revealed a protein of 704 amino acids(23) . Rabphilin3a is a
peripheral membrane protein (22, 24) which is composed
of at least two functionally distinct domains: the amino-terminal
region (1-280 amino acids) which binds to Rab3a and the
carboxyl-terminal domain (281-704 amino acids) which contains two
C2 domains that confer Ca
Bovine chromaffin cells are primary,
nondividing secretory cells that store and secrete catecholamine and a
variety of proteins including chromogranins, opiate peptides, and
opiate precursors. Secretion is normally stimulated by an influx of
Ca
Immunocytochemistry to detect human
GH and the HA1 epitope was performed as described
previously(16) .
Human
growth hormone was measured with a high sensitivity, chemiluminescence
assay kit from Nichols Institute (San Juan Capistrano, CA). Endogenous
catecholamine secretion was measured with a fluorescence
assay(33) . Secretion was expressed as the percentage of the
total cellular human GH or catecholamine that was released into the
medium. There was usually 0.2-1 ng of GH and 80-100 nmol of
catecholamine/35-mm diameter dish.
The
nucleotide sequence of c-Rabphilin3a is identical with that of
b-Rabphilin3a except for an 18-bp insert in the coding region (Fig. 1) and an A for T substitution 20 nucleotides upstream from
the ATG start codon. The insertion site starts with the consensus GT
sequence for splicing suggesting that the chromaffin cell and brain
forms of Rabphilin3a result from alternative splicing. The predicted
amino acid sequence of c-Rabphilin3a is identical with that of
b-Rabphilin3a except for the 6-amino-acid insert VFSLSA (encoded by the
18-bp insertion) following Arg-206 in the amino-terminal half of the
protein (underlined in Fig. 1). This insert may be a
phosphorylation site although a specific protein kinase is not
suggested by the sequence or the neighboring amino acids.
The effects on secretion of transfection
with plasmids encoding bovine adrenal chromaffin cell Rabphilin3a
(pCMV-HAcRP) and bovine brain Rabphilin3a (pCEV4-Rabphilin3a) were
examined. Expression of both b-Rabphilin3a and c-Rabphilin3a (Fig. 3) enhanced DMPP (20 µM)-induced secretion.
Expression of pRK7-cRP (non-epitope-tagged c-Rabphilin3a) enhanced
secretion similarly (data not shown). The effects were observed in 10
of 10 experiments with average enhancements of secretion of 34 ±
6% and 32 ± 10% between 0-2 min and 2-16 min,
respectively (p < 0.001 during each time interval versus transfection with pCMV.neo). The enhancement of
secretion by c- and b-Rabphilin3a is a specific effect of expression of
the protein since expression of numerous other exogenous proteins
including
Previous studies demonstrating an inhibitory role of Rab3a in
Ca
A comparison of the predicted
amino acid sequences of bovine brain (23), rat brain(22) , and
mouse (36) Rabphilin3a indicates that the protein is highly
conserved across species. The corresponding cysteine-rich region in the
amino-terminal region and the C2 domains in the carboxyl-terminal
region are at least 98% identical. Thus, the biochemical
characteristics of the proteins from the different species are likely
to be similar, and the highly conserved regions may be important for
the physiological function of the protein. Indeed, as discussed below,
both C2 domains are necessary for Rabphilin3a function in secretion.
A sequential model for
Rab3a-Rabphilin3a interaction is also consistent with the inability of
expression of either Rab3aT36N (16, 17) or
Rab3a
C2
domains may regulate Ca
We are grateful for the ongoing discussions about this
work with Dr. Ian G. Macara (Dept. of Pathology, University of Vermont
Medical School, Burlington, VT), Dr. Mary A. Bittner (Dept. of
Pharmacology, University of Michigan Medical School), and Dr. Michael
D. Uhler (Dept. of Biological Chemistry and the MHRI, University of
Michigan).
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
-dependent
secretion from digitonin-permeabilized cells. These data indicate that
Rabphilin3a is a positive regulator of exocytosis. Because the C2
deletion mutants contain the amino-terminal Rab3a-GTP binding domain,
they may inhibit secretion by competing with endogenous Rabphilin3a for
interaction with Rab3a-GTP without being able to mimic the functional
effects of full-length Rabphilin3a.
(
)complex and includes the vesicle membrane proteins
VAMP and synaptotagmin, the plasma membrane proteins SNAP25 and
syntaxin, and the cytosolic proteins NSF and
- and
-SNAPs.
The SNARE hypothesis (4, 5, 6) proposes that
vesicle SNAREs interact specifically with cognate target SNAREs.
Docking and fusion would proceed as a consequence of sequential
interactions modulated by NSF.
-dependent secretion from
permeabilized chromaffin cells(16) . Since the initial secretory
rates reflect secretion that is already primed by the previous action
of ATP (18, 19), overexpression of Rab3a may reduce the number of
chromaffin granules in the readily releasable pool.
-dependent lipid
binding(25) . The interaction of Rabphilin3a with Rab3a inhibits
GTPase-activating protein activity (26) and enhances guanine
nucleotide exchange(27) . These effects should maintain Rab3a in
the GTP-bound state within the cell. Rab3a and Rabphilin3a are both
localized to synaptic vesicles (22, 24, 28, 29) where they may
interact. Removal of the Rab3a gene in mice results in loss of
Rabphilin3a in brain suggesting that Rab3a stabilizes
Rabphilin3a(30) . These data support the notion that Rabphilin3a
is an effector for Rab3a.
and occurs in permeabilized cells upon incubation
with micromolar and higher Ca
concentrations. In a
previous study we demonstrated that Rab3a can act as an inhibitory
modulator of secretion in bovine chromaffin cells by using a transient
transfection technique. A plasmid of interest was co-transfected with a
plasmid encoding human growth hormone (GH), a reporter for the
regulated secretory pathway. Although only approximately 0.1% of the
cells were transfected, a sensitive immunological assay for GH could
readily detect its secretion(31) . Analysis of the effects of
various mutants suggested that the GTP-bound form of Rab3a was
responsible for the inhibition of secretion(16, 17) .
These results prompted us to investigate the role of Rabphilin3a in
secretion from the cells. In the present study, we isolated from a
bovine adrenal medullary cDNA library an apparent splice variant of the
bovine brain form of the protein and demonstrated that Rabphilin3a is
expressed in chromaffin cells. Using the transient transfection
approach, we provide the first evidence for a role of Rabphilin3a in
the regulated secretory pathway.
cDNA Cloning and Sequencing
cDNA encoding
Rabphilin3a from bovine adrenal medulla was identified on the basis of
its structural homology to bovine brain Rabphilin3a (23) using
PCR screening in a bovine adrenal medullary library pCDSP6T7 (from Dr.
Michael Brownstein, NIH). The nucleotide sequence of a cloned cDNA of
about 4.5 kb, containing a full-length coding sequence for chromaffin
cell Rabphilin3a (c-Rabphilin3a), was obtained.
Construction of Expression Vectors
The 4.5-kb BamHI fragment of c-Rabphilin3a was inserted into the BamHI site of the plasmid pRK7 to express Rabphilin3a cDNA
under the control of the CMV promoter (pRK7-cRP). Other cDNA constructs
were made by the polymerase chain reaction (PCR) using specific
oligonucleotide primers to generate sense (amino acids 1-710;
pCMV-HAcRP), antisense(1-710), and deletion mutant constructs
(1-286, 1-536, and 287-710) of c-Rabphilin3a. The
constructs were inserted into a plasmid under the control of the CMV
promoter and containing the HA1 epitope (YPYDVPDYA) in-frame at the
amino terminus. The construction of the mammalian expression vector for
bovine brain Rabphilin3a, pCEV4-Rabphilin3a, has been described
previously(23) .
Generation of Anti-Rabphilin3a Antibody
A rabbit
antibody against the 16 carboxyl-terminal amino acids
(IERWHQLQNENHVSSD) common to both the chromaffin cell and brain forms
of Rabphilin3a was generated from a KLH-peptide immunogen emulsified by
mixing with Freund's adjuvant. High titers of antibody were
obtained at 10 and 14 weeks as determined by an enzyme-linked
immunosorbent assay against the peptide.
Protein Expression and Immunocytochemistry
Protein
expression was examined by transient transfection of COS7 cells in
35-mm diameter dishes by calcium phosphate precipitation. After 3 days,
cells were harvested into sample buffer and separated on 5-15%
gradient SDS-polyacrylamide gel electrophoresis and transferred to
Immobilon. The blot was incubated sequentially with a rabbit
anti-Rabphilin3a antiserum (1:500 dilution) and I-goat
anti-rabbit antibody. Chromaffin granule membranes were purified from a
P2 fraction from fresh bovine adrenal medulla by centrifugation through
1.7 M sucrose(32) . The chromaffin granule pellet was
resuspended in hypotonic solution, and the chromaffin granule membranes
were pelleted by centrifugation.
Cell Preparation, Transfection, and Secretion
Experiments
Chromaffin cell preparation, transient transfection,
and secretion experiments were performed as described
previously(16, 31) . Secretion experiments were
performed 5-6 days after the transfection at 27 °C.
Isolation of Rabphilin3a cDNA Clone from Bovine
Chromaffin Cells
A cDNA encoding Rabphilin3a from bovine adrenal
medulla was identified on the basis of its structural homology to
bovine brain Rabphilin3a (b-Rabphilin3a) using PCR screening in a
bovine adrenal medullary library pCDSP6T7. The nucleotide sequence of a
cloned cDNA of about 4.5 kb was determined and contained a full-length
coding sequence for chromaffin cell Rabphilin3a (c-Rabphilin3a).
Figure 1:
Chromaffin cell Rabphilin3a is a likely
splice variant of brain Rabphilin3a. The nucleotide sequence of a clone
from a chromaffin cell c-DNA library was identical with that of brain
Rabphilin3a (23) except for an 18-bp insert in the coding region and an
A for T substitution 20 nucleotides upstream from the ATG start codon.
Shown are the nucleotide and predicted amino acid sequences for brain
and chromaffin cell Rabphilin3a in the coding region that encompasses
the insert (underlined). The numbering corresponds to
the amino acid number in the sequence.
Expression of Rabphilin3a in COS7 Cells and Chromaffin
Granules
We constructed a plasmid encoding c-Rabphilin3a under
the control of the CMV promoter (pCMV-HAcRP). COS7 cells were
transfected with a plasmid encoding either the brain form of
Rabphilin3a (pCEV4-Rabphilin3a) or the chromaffin cell form of
Rabphilin3a (pCMV-HAcRP). The anti-Rabphilin3a antibody recognized a
protein at 85 kDa in COS7 cells transfected with plasmids encoding b-
and c-Rabphilin3a (Fig. 2A, lanes B and C, respectively) but not in the cells transfected with the
control plasmid (Fig. 2A, lane D). These
experiments indicate that the cloned cDNA from bovine chromaffin cells
encodes a protein of appropriate molecular weight for Rabphilin3a. The
antibody also recognized a protein of similar molecular weight in a
highly purified chromaffin granule membrane preparation (Fig. 2A, lane A).
Figure 2:
Expression of transiently transfected
plasmid encoding Rabphilin3a in COS7 cells and in bovine adrenal
chromaffin cells. A, antibody against the carboxyl-terminal
domain of Rabphilin3a (see ``Materials and Methods'')
identifies Rabphilin3a in chromaffin granules and in COS7 cells
expressing cDNA encoding the brain and chromaffin cell isoforms in
immunoblots. Lane A, highly purified chromaffin granule
membranes (50 µg of protein); B, COS7 cells (7.5 µg of
protein) 72 h after transfection with plasmid encoding brain
Rabphilin3a; C, COS7 cells (7.5 µg of protein) 72 h after
transfection with a plasmid encoding chromaffin cell Rabphilin3a; D, COS7 cells (7.5 µg of protein) 72 h after transfection
with control plasmid. The plasmid encoding c-Rabphilin3a was under the
control of the CMV promoter; the plasmid encoding b-Rabphilin3a was
under the control of the SR promoter. The antibody recognizes an
85-kDa protein (indicated as Rabphilin3a) in COS7 cells transfected
with the plasmid encoding either brain or chromaffin cell Rabphilin3a (lanes B and C) and in chromaffin granule membranes (lane A). Preimmune antibody did not detect the band. B, HA1-tagged Rabphilin3a derived from an adrenal medullary
library can be co-expressed with human GH in chromaffin cells. Bovine
chromaffin cells were co-transfected with pXGH5 encoding human GH and
pCMV-HAcRP encoding HA1-tagged c-Rabphilin3a. Five days later, cells
were fixed and permeabilized with methanol. Rabbit anti-human GH (with
donkey anti-rabbit lissamine rhodamine) and mouse monoclonal anti-HA1
(12CA5, with goat anti-mouse fluorescein isothiocyanate) was used to
detect GH (middle) and the HA1-tagged c-Rabphilin3a (right). The phase image of the field is on the left.
Overexpression of c- and b-Rabphilin3a Enhances Secretion
from Chromaffin Cells
We investigated the effects on GH
secretion of co-transfection with plasmids encoding Rabphilin3a and
pXGH5 (encoding human GH). To determine whether the transfected
Rabphilin3a is co-expressed with human growth hormone in chromaffin
cells, immunocytochemistry was performed on chromaffin cells. Plasmid
encoding c-Rabphilin3a (pCMV-HAcRP) was co-transfected with pXGH5. An
epitope tag (HA1) was attached to the amino terminus of the Rabphilin3a
protein to allow detection in the cells. Fig. 2B demonstrates that Rabphilin3a could be co-expressed with human GH
when it was co-transfected with pXGH5 in chromaffin cells.
Approximately 90% of the growth hormone-expressing cells expressed
detectable amounts of epitope-tagged protein as determined by
fluorescence microscopy.
-galactosidase, certain mutants of Rab3a (Rab3a
CAC
and Rab3aV55E), and Rabin3a do not alter GH secretion in transfected
chromaffin cells. The latter protein, Rabin3a, was recently cloned from
a rat brain cDNA library as a Rab3a-binding protein using the yeast
two-hybrid system(34) .
Figure 3:
Expression of brain Rabphilin3a and
chromaffin cell Rabphilin3a enhances secretion from chromaffin cells.
Chromaffin cells were co-transfected with pXGH5 encoding human GH and
either pCEV4-Rabphilin3a encoding bovine brain Rabphilin3a or
pCMV-HAcRP encoding HA1-tagged bovine chromaffin cell-Rabphilin3a. Six
days later, cells were incubated in physiological salt solution for 2
min ± 20 µM DMPP. There were 4 dishes/group. a, p < 0.05 versus GH secretion from
cells transfected with control plasmid,
pCMV.neo.
Transfection with the Antisense Construct of
c-Rabphilin3a Inhibits Secretion from Chromaffin Cells
Since
overexpression of Rabphilin3a enhances secretion, reduced expression of
endogenous Rabphilin3a by transfection with an antisense Rabphilin3a
construct should reduce stimulated secretion. Fig. 4suggests
that this is, indeed, the case. Chromaffin cells were transfected with
a plasmid with the entire coding sequence of c-Rabphilin3a inserted in
the reverse orientation in the expression vector downstream from a CMV
promoter. Six days later, DMPP-induced secretion was inhibited by
approximately 30% (Fig. 4). In four experiments, the average
inhibition of secretion between 0-2 min and 2-16 min was 27
± 4% during each time interval (p < 0.001 during
each time interval, compared to transfection with pCMV.neo).
Figure 4:
Transient transfection with antisense
Rabphilin3a inhibits secretion of co-transfected and expressed GH. GH
secretion was determined from chromaffin cells that were co-transfected
with the plasmid encoding human GH (pXGH5) and either a plasmid
constructed with the entire coding region for chromaffin cell
Rabphilin3a in the antisense orientation (Anti-Sense) or with
the parent plasmid (CMV.neo). Six days after transfection,
cells were incubated with and without 20 µM DMPP for 2 min
in physiological salt solution, and DMPP-dependent secretion was
determined. There were 7-8 dishes/groups. a, p < 0.01 versus pCMV.neo.
Effects of Deletion Mutants of Rabphilin3a on
Secretion
Chromaffin cells were co-transfected with pXGH5 and
several deletion mutants of Rabphilin3a (Fig. 5A). The
constructs lacking one (RpC2B) or both (Rp
C2AB) of the
carboxyl-terminal C2 domains but containing the Rab3a binding domain of
the amino-terminal portion of the Rabphilin3a strongly inhibited
secretion in the first 2 min. The construct without the Rab3a binding
domain, Rp(287-710), was without effect although
immunocytochemistry revealed that the transfected mutant was
co-expressed with human growth hormone in chromaffin cells (data not
shown). Truncated Rabphilin3a lacking one or both C2 domains retains
the amino-terminal sequence necessary for binding Rab3a-GTP and is
likely to bind Rab3a-GTP similarly to the full-length protein. Thus,
the results with the deletion mutants raise the possibility that
truncated Rabphilin3a competitively inhibits the function of endogenous
Rabphilin3a.
Figure 5:
Effects of Rabphilin3a mutants on
secretion from intact (A) and permeabilized (B)
cells. Chromaffin cells were co-transfected with pXGH5 encoding human
GH and either pCMV.RpC2B encoding Rabphilin3a(1-536) without
the second C2 domain, pCMV.Rp
C2AB encoding
Rabphilin3a(1-286) without both C2 domains,
pCMV.Rp(287-710) encoding just the carboxyl-terminal half of the
protein, or pCMV.neo, the parent plasmid. Six days later, cells were
incubated in physiological salt solution ± 20 µM DMPP. DMPP-induced GH secretion was determined between 0-2
min and 2-16 min (A) or were permeabilized for 4 min
with digitonin-containing potassium glutamate solution with and without
30 µM Ca
(B). There were four
35-mm diameter dishes/group. a, p < 0.01 versus pCMV.neo.
One of the mutants (RpC2B) was further
investigated in permeabilized cells (Fig. 5B). Its
expression strongly inhibited Ca
-dependent secretion.
Thus, it is likely that the inhibition was a direct effect on the
Ca
-dependent secretory pathway and not on the
Ca
signal.
-dependent exocytosis in bovine chromaffin cells
prompted us to investigate the function of the Rab3a-binding protein,
Rabphilin3a, as a possible effector for Rab3a function. The present
study identified Rabphilin3a in bovine adrenal chromaffin cells,
probably associated with chromaffin granules. Overexpression of
Rabphilin3a by transient transfection of the cDNA enhanced secretion,
and transfection with antisense Rabphilin3a cDNA inhibited secretion.
The data suggest that Rabphilin3a is likely to be a positive regulator
of secretion.
Bovine Chromaffin Cells Express a Splice Variant of the Bovine
Brain Rabphilin3a
The predicted amino acid sequence of the
Rabphilin3a cDNA clone identified in a bovine adrenal medullary cDNA
library was identical with that of bovine brain Rabphilin3a except for
a 6-amino-acid insert VFSLSA (encoded by the 18-bp insertion) following
Arg-206 in the amino-terminal half of the protein (underlined in Fig. 1). The insert probably resulted from alternative
splicing of the mRNA. Both forms of Rabphilin3a enhanced secretion from
bovine chromaffin cells (Fig. 3). Alternative splicing probably
also occurs in rat brain Rabphilin3a (22) in which there is a
3-amino-acid insert in the amino-terminal region. The expression of
Rabphilin3a was confirmed by protein immunoblots. These results,
together with the identification of Rabphilin3a in PC12
cells(35, 36) and mRNA analysis in an insulin-secreting
cell line (HIT-T15 cells), suggest a function for Rabphilin3a in
endocrine cells as well as in brain.
Opposite Effects on Secretion of Rab3a and Rabphilin3a
Suggest a Model with Sequential Events
The high affinity and
specific binding of Rab3a-GTP with Rabphilin3a in
vitro(20, 23) , the association of both Rab3a and
Rabphilin3a with synaptic vesicles and probably chromaffin granules,
and the evidence for Rab3a-Rabphilin3a interaction in vivo(30) suggest that the function of these proteins in
secretion is likely to be governed by their interaction in the cell. If
the inhibitory effect of overexpression of Rab3a on secretion were
directly mediated by its interaction with Rabphilin3a, then
overexpression of Rabphilin3a should have also inhibited secretion. The
opposite effects of Rab3a to inhibit and of Rabphilin3a to enhance
secretion indicate that the functional relationship between the
proteins is probably more complex. One explanation that is consistent
with the data is that there is a sequence of events in which the
formation of Rab3a-GTPRabphilin3a complex is necessary for
secretion but the complex must dissociate (perhaps because of GTP
hydrolysis) in order for secretion to occur. Free, activated
Rabphilin3a would be a positive effector for secretion. Overexpression
of Rab3a would inhibit secretion by rapidly rebinding uncomplexed
Rabphilin3a. The model is compatible with the inhibition of secretion
by expression of the Rabphilin3a deletion mutants without one or both
of the C2 domains. Because these mutants bind Rab3a-GTP, they may
inhibit secretion by competing with endogenous Rabphilin3a for
interaction with Rab3a-GTP without being able to mimic the functional
effects of full-length Rabphilin3a. Reduced binding of endogenous
Rabphilin3a with Rab3a may also result in increased degradation of
either or both endogenous Rabphilin3a and
Rab3a(22, 30) .
CAC
(
)to inhibit secretion. Rab3aT36N
does not alter secretion because it is predominantly in the GDP-bound
state that does not bind Rabphilin3a(21) ; unprenylated
Rab3a
CAC does not alter secretion because it does not bind to
membranes.
The Presence of Both C2 Domains Is Necessary for Wild
Type Rabphilin3a Function
The strong inhibition of secretion by
expression of Rabphilin3a mutants without one or both of the C2 domains
contrasts with the enhancement of secretion by overexpression of
full-length Rabphilin3a. The results provide direct evidence for the
importance of both of the C2 domains for the physiological
function of Rabphilin3a. Both C2 domains are also necessary for
synaptotagmin function(37) . A single C2 domain is sufficient to
convey Ca-dependent effects to protein kinase C (38) and phospholipase A2(39) . Model studies with single
C2 domains of synaptotagmin also demonstrate
Ca
-dependent lipid binding(40, 41) .
The tandem C2 domains of Rabphilin3a (and of synaptotagmin) may confer
specificity of binding to target proteins or permit the simultaneous
binding to different membranes or membrane proteins. Such effects could
be important in positioning the granule at the plasma membrane.
/phospholipid-dependent
binding to specific proteins(42) . Indeed, it has been reported
that Rabphilin3a interacts with
-adducin in a
Ca
- and phospholipid-dependent manner(43) .
-Adducin is associated with the subplasmalemmal cytoskeleton and
probably catalyzes cytoskeleton assembly(44) . It is tempting to
speculate that Rabphilin3a bound to the chromaffin granule membrane may
reduce the cytoskeletal barrier for granule interaction with the plasma
membrane by inhibiting
-adducin function.
Rab3a, Rabphilin3a, and SNAREs
Although at this
time direct physical interaction of Rab proteins with SNARE proteins
has not been demonstrated, genetic and biochemical evidence in yeast
suggest that the Rab pathway interacts in some manner with the
v-SNARE/t-SNARE pathway(45, 46, 47) . To date, a
protein homologous to Rabphilin3a has not been identified in yeast.
However, it is possible that just as the Rab protein YPT1 enhances the
interaction of two v-SNAREs (Bos1p and Sec22p)(46) , Rab3a
enhances the functional interaction of Rabphilin3a with a SNARE as well
as with other proteins.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.