From the Department of Molecular Biology and Biochemistry, Osaka University Medical School, Suita 565, Japan
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ABSTRACT |
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Rabphilin3 and rabaptin5 are downstream target
molecules of the Rab3 and -5 subfamily small G proteins that are
implicated in exocytosis and endocytosis, respectively. We examined
here the physical and functional relationship between the
Rab3-rabphilin3 and Rab5-rabaptin5 systems. Rabphilin3 interacted with
rabaptin5 at the N-terminal region (amino acids 1-280), which
GTP-Rab3A interacted with. The interaction of rabphilin3 with rabaptin5 was inhibited by guanosine 5-(3-O-thio)triphosphate-Rab3A.
Overexpression of the N-terminal fragment of rabphilin3 (amino acids
1-280) inhibited the receptor-mediated endocytosis of transferrin, and
this inhibition was overcome by co-transfection with a dominant active
mutant of Rab3A or rabaptin5 in PC12 and HeLa cells. These results
suggest that rabphilin3, free of GTP-Rab3A, regulates endocytosis
through interaction with rabaptin5 after rabphilin3 complexed with
GTP-Rab3A regulates exocytosis.
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INTRODUCTION |
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Endocytosis is often coupled with exocytosis, particularly at nerve terminals (for reviews, see Refs. 1 and 2). Molecular mechanisms of exocytosis and endocytosis have extensively been investigated, but the coupling mechanism between these two events is unknown. The Rab family small G proteins are implicated in intracellular vesicle trafficking, such as exocytosis and endocytosis (for reviews, see Refs. 3-5). The Rab3 subfamily consists of four members, Rab3A, -B, -C, and -D, and Rab3A and -C are implicated in Ca2+-dependent exocytosis, particularly in neurotransmitter release from nerve terminals (for a review, see Ref. 6). A current model for the mode of action of Rab3A and -C in neurotransmitter release is as follows: GDP-Rab3 complexed with Rab GDI1 stays in the presynaptic cytosol and is converted to GTP-Rab3 by the action of Rab3 GEP. GTP-Rab3 interacts with rabphilin3 associated with synaptic vesicles. This interaction causes the docking of the vesicles with the presynaptic plasma membrane by an unknown mechanism. Ca2+ influx into the cytosol causes the fusion of the vesicles with the presynaptic plasma membrane, finally resulting in neurotransmitter release. In one of these processes, GTP-Rab3 is converted to GDP-Rab3 by the action of Rab3 GAP. GDP-Rab3 is complexed with Rab GDI and returns to the cytosol. In this Rab3 system, we have isolated and characterized Rab3A, -B, -C, Rab GDI, rabphilin3, Rab3 GEP, and Rab3 GAP (7-14). The Rab5 subfamily consists of three members, Rab5A, -B, and -C, and is implicated in endocytosis (3, 15-18). A model for the mode of action of Rab5 in endocytosis is as follows: GDP-Rab5 complexed with Rab GDI stays in the cytosol and is converted to GTP-Rab5 by the action of Rab5 GEP after its association with early endosomes. GTP-Rab5 interacts with rabaptin5 and recruits it to early endosomes. This interaction causes both the fusion of the plasma membrane-derived endocytotic vesicles with early endosomes and the lateral fusion of early endosomes. In one of these processes, GTP-Rab5 is converted to GDP-Rab5 by the action of Rab5 GAP. GDP-Rab5 is complexed with Rab GDI and returns to the cytosol. In this Rab5 system, Rab5, rabaptin5, and Rab5 GAP have been isolated and characterized (15, 18, 19), but Rab5 GEP has not yet been isolated or characterized.
In the Rab3-regulated exocytosis, it still remains unknown how the
interaction of Rab3 with rabphilin3 causes the docking of the vesicles
with the presynaptic plasma membrane. To clarify this mechanism, we
have attempted to isolate molecules interacting with rabphilin3.
Rabphilin3 consists of at least two functionally different domains: the
N-terminal C1-like domain responsible for interaction with GTP-Rab3,
and the C-terminal two C2-like domains responsible for interaction with
Ca2+ and phospholipid (20, 21). We have found that
-adducin, known to be involved in the assembly of spectrin-actin
complexes (for a review, see Ref. 22), interacts with the C-terminal
region of rabphilin3 in the presence of Ca2+ and
phospholipid (23, 24) and that
-actinin, known to cross-link actin
filaments into a bundle (for reviews, see Refs. 25 and 26), interacts
with the N-terminal region of rabphilin3 in a manner competitive with
GTP-Rab3 (27). Here, we have found that rabphilin3 interacts with
rabaptin5 at the N-terminal region and that this interaction is
inhibited by GTP-Rab3A. We have furthermore shown that rabphilin3 is
involved in endocytosis.
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EXPERIMENTAL PROCEDURES |
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Materials and Chemicals--
Bovine Rab3A and rabphilin3 were
purified from the membrane fraction of Spodoptera frugiperda
cells (Sf9 cells) overexpressing each protein (28, 29). GDP-Rab3A and
GTPS-Rab3A were prepared as described (30). Truncated rat rabaptin5,
which corresponds to an amino acid position from 385 to 753 of human
rabaptin5 (GenBank® accession number X91141), was expressed as a
GST-fusion protein and purified from overexpressing Escherichia
coli. Anti-HA and anti-myc mouse monoclonal antibodies and an
anti-rabphilin3 rabbit polyclonal antibody were prepared as described
(31-33).
Construction of Expression Vectors-- Mammalian expression plasmids, pEFBOS-HA and pEFBOS-myc, were generated to express fusion proteins with the N-terminal HA and myc epitopes, respectively (33, 34). Another mammalian expression plasmid, pGEM-HA, was generated to express a fusion protein with the N-terminal HA epitope, using the T7 RNA polymerase recombinant vaccinia virus (LO-T7) (35). The cDNA fragments encoding full-length rabphilin3 and its N-terminal fragment (1-280 aa) were inserted into pGEM-HA and pEFBOS-HA, respectively. The cDNA fragment encoding truncated rabaptin5 was inserted into pGEM-HA and pEFBOS-myc. The cDNA fragments encoding human transferrin receptor and a dominant active mutant of Rab3A (Rab3AQ81L) were inserted into pEFBOS-myc.
Strains and Media--
Yeast strain L40 (MATa trp1 leu2
his3 LYS2::lexA-HIS3 URA3::lexA-lacZ) was
grown on YPDAU medium that contained 2% glucose, 2% Bacto-peptone
(Difco), 1% Bacto-yeast extract (Difco), 0.04% adenine sulfate, and
0.02% uracil. Yeast transformations were performed by the lithium
acetate methods (36). Transformants were selected on SD medium that
contained 2% glucose and 0.7% yeast nitrogen base without amino
acids, and amino acids (Difco) were supplemented to SD medium when
required. Standard yeast genetic manipulations were performed as
described (37). An E. coli strain DH5 was used for
construction and propagation of plasmids.
Screening for Rabphilin3-interacting Molecules by the 2-hybrid
Method--
A strain L40 was transformed with a derivative of pBTM116
bearing the N-terminal fragment (1-280 aa) of rabphilin3 fused to the
LexA DNA-binding domain: pBTM116-rabphilin3-N. A strain L40 carrying
pBTM116-rabphilin3-N was transformed with the library DNA (MATCHMAKER
rat brain oligo(dT)-primed library in pGAD10, CLONTECH). Approximately 1.1 × 106 transformants were screened for the growth on SD plate
media lacking tryptophan, leucine, and histidine, but containing 0.5 mM 3-amino-1,2,4-triazole, which is a specific inhibitor of
the HIS3 gene product. His+ colonies were then
placed on the nitrocellulose filter and stained with
5-bromo-4-chloro-3-indolyl--D-galactopyranoside for
-galactosidase activity as described (38). From the 50 His+ and lacZ+ positive clones obtained with
this screening, library plasmids were recovered through E. coli transformation. The recovered plasmids were transformed again
into L40 containing pBTM116-rabphilin3-N, and it was found that 14 clones conferred the His+ and lacZ+ phenotypes
on L40 containing pBTM116-rabphilin3-N. The nucleotide sequences of the
insert DNA of these 14 clones were determined.
Assay for Rabphilin3-Rabaptin5 Interactions in an Intact Cell System-- PC12 cells were plated at a density of 1.5 × 106 cells/35-mm dish and then incubated for 18 h at 37 °C. The cells were infected with T7 RNA polymerase recombinant vaccinia virus (LO-T7) for 30 min and then transfected with 2 µg of pGEM-HA-rabaptin5, 2 µg of pGEM-HA-rabphilin3, or both the plasmids, by use of LipofectAMINE reagent (Life Technologies, Inc.) (35). Immunoprecipitation was performed at 4 h after the transfection. The cells were washed with phosphate-buffered saline and lysed in 0.5 ml per dish of a lysis buffer containing 10 mM Tris/HCl at pH 7.5, 150 mM NaCl, 1.5% Nonidet P-40, 91 µM CaCl2, and 1 mM EGTA. The cell lysate was subjected to immunoprecipitation with 10 pmol of the anti-rabphilin3 antibody bound to 25 µl of protein A-Sepharose (Pharmacia Biotech Inc.). Comparable amounts of the pellets were subjected to SDS-PAGE, followed by immunoblot analysis with the anti-HA antibody.
Assay for Rabphilin3-rabaptin5 Interactions in a Cell-free
System--
Full-length rabphilin3 (50 pmol) was incubated with GST or
GST-rabaptin5 (100 pmol) bound to glutathione-Sepharose in a buffer containing 20 mM Tris/HCl at pH 7.5, 1 mM
dithiothreitol, 5 mM MgCl2, 0.5% Nonidet P-40,
and 20 mM NaCl for 90 min at 4 °C in the presence or
absence of 0.4 µM GDP-Rab3A or GTPS-Rab3A. After the
beads were washed four times with the same buffer, the bound proteins
were eluted by addition of 100 µl of a buffer containing 20 mM Tris/HCl at pH 8.0, 1 mM dithiothreitol, 5 mM MgCl2, 0.5% Nonidet P-40, 20 mM
NaCl, and 10 mM reduced glutathione. The eluates were
subjected to SDS-PAGE, followed by protein staining with Coomassie
Brilliant Blue.
Assay for the Receptor-mediated Endocytosis in PC12 and HeLa Cells-- PC12 or HeLa cells were transfected with the plasmids encoding the indicated proteins by use of Lipofectin reagent (Life Technologies, Inc.). After 48 h, the cells were incubated for 60 min at 37 °C with serum-free Dulbecco's modified Eagle's medium containing 0.1 mg/ml FITC-transferrin (Molecular Probes, Inc.). The cells were then fixed with 4% formaldehyde and permeabilized with 0.2% Triton X-100. The HA and myc epitopes were detected with the anti-HA and anti-myc antibodies as the first antibodies, respectively, and rhodamine-conjugated anti-mouse donkey antibody (Chemicon International) as the second antibody. To examine the co-expression of the HA- and myc-tagged proteins, rhodamine-conjugated anti-HA antibody and FITC-conjugated anti-myc antibodies were used. The cells were analyzed with a LSM 410 confocal laser scanning microscope (Carl Zeiss, Oberkochen, Germany). To examine whether the expression level of the indicated protein was affected by co-expression of the other proteins, transfected HeLa cells were lysed, and the expression levels of the HA-tagged and myc-tagged proteins were examined by immunoblot analysis of whole cell lysates using the anti-HA and anti-myc antibodies, respectively.
Other Procedures-- SDS-PAGE was performed as described (39). Rabphilin3 and rabaptin5 transferred to nitrocellulose sheets were detected using the ECL immunoblotting detection system (Amersham Corp.). Protein concentrations were determined with bovine serum albumin as a standard protein (40).
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RESULTS |
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Interaction of Rabphilin3 with Rabaptin5 Estimated by the Yeast two-Hybrid System-- We first attempted to isolate rabphilin3-interacting proteins by use of the yeast two-hybrid system with the N-terminal fragment or the C-terminal fragment of rabphilin3 as a bait from a rat brain cDNA library. We did not isolate any clones with the C-terminal fragment as a bait. But with the N-terminal fragment as a bait, we isolated several clones and selected one clone (N22) encoding a protein, which corresponds to an amino acid position from 385 to 753 of human rabaptin5, because rabaptin5 is known to be the downstream target molecule of the Rab5 subfamily implicated in endocytosis (3, 15-18). The isolated clone indeed interacted with the N-terminal fragment of rabphilin3 by the yeast two-hybrid method, and this interaction was comparable to the interaction of Rab3A with rabphilin3 (Fig. 1A). This clone also interacted with full-length rabphilin3 but not with its C-terminal fragment (Fig. 1B).
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Rabphilin3-Rabaptin5 Interactions in an Intact Cell System-- The rabphilin3-rabaptin5 interactions were confirmed by co-immunoprecipitation of HA-tagged full-length rabphilin3 and HA-tagged truncated rabaptin5 from cultured PC12 cells. PC12 cells were transiently transfected with the plasmid encoding HA-tagged rabphilin3 or HA-tagged rabaptin5, or with both the plasmids. The cells were lysed in the lysis buffer, and the cell lysates were incubated with the anti-rabphilin3 antibody immobilized on protein A-Sepharose. Rabphilin3 was immunoprecipitated by the anti-rabphilin3 antibody in both the cells expressing HA-tagged rabphilin3 alone and the cells expressing HA-tagged rabphilin3 and HA-tagged rabaptin5 (Fig. 2, lanes 2 and 3). Rabaptin5 was not co-immunoprecipitated by the anti-rabphilin3 antibody in the cells expressing HA-tagged rabaptin5 alone or the cells expressing HA-tagged rabphilin3 alone (Fig. 2, lanes 1 and 2) but co-immunoprecipitated with rabphilin3 in the cells expressing both HA-tagged rabphilin3 and HA-tagged rabaptin5 (Fig. 2, lane 3).
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Inhibition by GTPS-Rab3A of the Rabphilin3-Rabaptin5
Interactions--
The rabphilin3-rabaptin5 interactions were further
confirmed by use of the recombinant proteins. GST-rabaptin5 was
prebound to glutathione-Sepharose, and the binding of rabphilin3 to
this prebound protein was analyzed. Rabaptin5 indeed interacted with rabphilin3 (Fig. 3, lanes 1 and 2).
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Inhibition of the Receptor-mediated Endocytosis of Transferrin by Rabphilin3-- Finally, effect of rabphilin3 on the receptor-mediated endocytosis of transferrin was examined. PC12 cells were transiently transfected with the plasmid encoding myc-tagged human transferrin receptor alone or co-transfected with the plasmid encoding the HA-tagged N-terminal fragment of rabphilin3 and then incubated with FITC-transferrin. The expressed myc-tagged human transferrin receptor and HA-tagged N-terminal fragment of rabphilin3 were detected by confocal immunofluorescence microscopy using the anti-myc and anti-HA antibodies, respectively. The FITC-transferrin incorporated into the cells by endocytosis was examined by confocal immunofluorescence microscopy. In the cells expressing myc-tagged human transferrin receptor alone, FITC-transferrin was markedly incorporated into the cells (Fig. 4A, 1 and 2). However, in the cells co-expressing both myc-tagged human transferrin receptor and the HA-tagged N-terminal fragment of rabphilin3, the incorporation of FITC-transferrin into the cells was not detected (Fig. 4A, 3 and 4). This inhibitory action of the N-terminal fragment of rabphilin3 was overcome by co-expression with a dominant active mutant of Rab3A (Rab3AQ81L) (Fig. 4A, 5 and 6) or rabaptin5 (Fig. 4A, 7 and 8). These data were obtained in about 90% of the cells expressing the HA-tagged N-terminal fragment of rabphilin3. The co-expression of the HA-tagged N-terminal fragment of rabphilin3 and myc-tagged human transferrin receptor was confirmed by the anti-HA and anti-myc antibodies, respectively (Fig. 4A, 9 and 10). The expression of the Rab3A mutant and rabaptin5 was confirmed by confocal immunofluorescence microscopy using the anti-myc antibody (data not shown).
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DISCUSSION |
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We have shown here by use of three different methods, the yeast two-hybrid, affinity column chromatography, and immunoprecipitation, that rabphilin3 directly interacts with rabaptin5. Rabaptin5 interacts with the N-terminal region of rabphilin3, and this interaction is inhibited by GTP-Rab3A. The precise mode of action of the Rab3-rabphilin3 system in Ca2+-dependent neurotransmitter release has not been clarified, but GTP-Rab3 is converted to GDP-Rab3 by the action of Rab3 GAP before, during, or after the fusion of synaptic vesicles with the presynaptic plasma membrane (6). Our results suggest that rabphilin3 interacts with rabaptin5 after GTP-Rab3 is converted to GDP-Rab3 and released from rabphilin3.
Moreover, we have shown here that overexpression of the N-terminal fragment of rabphilin3 in PC12 and HeLa cells inhibits the receptor-mediated endocytosis as estimated by the incorporation of FITC-transferrin and that this inhibition is overcome by co-expression with the dominant active mutant of Rab3A or rabaptin5. These results suggest that the N-terminal fragment interacts with endogenous rabaptin5 and inhibits its function in the endocytotic processes. The dominant active mutant of Rab3A or rabaptin5 may overcome the inhibitory action of the N-terminal fragment of rabphilin3 by inhibiting the interaction of the N-terminal fragment with endogenous rabaptin5. These results are consistent with the fact that rabphilin3 free of GTP-Rab3A, not that complexed with GTP-Rab3A, interacts with rabaptin5.
Endocytosis is often coupled with exocytosis, especially at nerve terminals (1, 2). The mechanism of this type of endocytosis remains a matter of debate, but several lines of evidence implicate that endocytosis of synaptic vesicles appears to resemble receptor-mediated endocytosis mediated by clathrin-coated vesicles in non-neuronal cells (for a review, see Ref. 41). However, since endocytosis of synaptic vesicles is faster than receptor-mediated endocytosis in non-neuronal cells and is not inhibited by decrease in the pH or the K+ concentration which disrupt receptor-mediated endocytosis (42), endocytosis of synaptic vesicles is considered a specialization of receptor-mediated endocytosis. Therefore, our present results used here indicate that the Rab3-rabphilin3 system is involved in receptor-mediated endocytosis, but it is not known whether rabphilin3 is implicated in endocytosis coupled with Ca2+-dependent exocytosis from nerve terminals. Further study is necessary for the precise role of rabphilin3 in endocytosis.
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ACKNOWLEDGEMENTS |
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We thank Dr. Michinori Kohara (The Tokyo Metropolitan Institute of Medical Science, Tokyo), Dr. Shigekazu Nagata (Osaka University Medical School, Suita), and Dr. Satoshi Orita (Shionogi Institute for Medical Science, Settsu) for providing the T7 RNA polymerase recombinant vaccinia virus (LO-T7), plasmid pEF-BOS, and the cDNA of human transferrin receptor, respectively.
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FOOTNOTES |
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* This work was supported by grants-in-aid for Scientific Research and for Cancer Research from the Ministry of Education, Science, Sports, and Culture, Japan (1996, 1997), by grants-in-aid for Abnormalities in Hormone Receptor Mechanisms and for Aging and Health from the Ministry of Health and Welfare, Japan (1996, 1997), and by grants from the Human Frontier Science Program (1996, 1997) and the Uehara Memorial Foundation (1996).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Present address: The Third Department of Internal Medicine,
Faculty of Medicine, Kyushu University, Fukuoka 812-82, Japan.
§ To whom correspondence should be addressed. Tel.: 81-6-879-3410; Fax: 81-6-879-3419; E-mail: ytakai{at}molbio.med.osaka-u.ac.jp.
1
The abbreviations used are: GDI, GDP
dissociation inhibitor; GEP, GDP/GTP exchange protein; GAP, GTPase
activating protein; GTPS, guanosine
5
-(3-O-thio)triphosphate; HA, hemagglutinin; aa, amino
acid; SD, synthetic dextrose; PAGE, polyacrylamide gel electrophoresis;
FITC, fluorescein isothiocyanate.
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REFERENCES |
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