From the Membrane Biology Laboratory, Institute of Molecular and Cell Biology, Singapore 117609, Singapore
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ABSTRACT |
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Expressed sequence tags coding for a potential
SNARE (soluble N-ethylmaleimide-sensitive factor attachment
protein receptor) were revealed during data base searches. The deduced
amino acid sequence of the complete coding region predicts a
217-residue protein with a COOH-terminal hydrophobic membrane anchor.
Affinity-purified antibodies raised against the cytoplasmic region of
this protein specifically detect a 29-kilodalton integral membrane
protein enriched in the Golgi membrane. Indirect immunofluorescence
microscopy reveals that this protein is mainly associated with the
Golgi apparatus. When detergent extracts of the Golgi membrane are
incubated with immobilized glutathione S-transferase soluble N-ethylmaleimide-sensitive factor attachment
protein (GST-
-SNAP), this protein was specifically retained. This
protein has been independently identified and termed Vti1-rp2, and it
is homologous to Vti1p, a yeast Golgi SNARE. We further show that
Vti1-rp2 can be qualitatively coimmunoprecipitated with Golgi syntaxin
5 and syntaxin 6, suggesting that Vti1-rp2 exists in at least two
distinct Golgi SNARE complexes. In cells microinjected with antibodies
against Vti1-rp2, transport of the envelope protein (G-protein) of
vesicular stomatitis virus from the endoplasmic reticulum to the plasma
membrane was specifically arrested at the Golgi apparatus, providing
further evidence for functional importance of Vti1-rp2 in protein
trafficking in the secretory pathway.
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INTRODUCTION |
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Participation of NSF1 and soluble NSF attachment proteins (SNAP) in diverse transport events in the secretory and endocytotic pathways is in conjunction with a superfamily of membrane proteins termed SNAP receptors (SNAREs) (1-5). The SNARE hypothesis suggests that vesicles derived from a donor compartment harbor a set of vesicle-associated SNAREs (v-SNAREs) that will interact specifically with those associated with those on the target acceptor membrane (t-SNAREs) (6-11). This v-/t-SNARE pairing is a key event in the docking and fusion of the vesicle with its specific target membrane (6-11). Vesicle-associated membrane proteins (VAMPs) or synaptobrevins are v-SNAREs associated with the synaptic vesicles, whereas syntaxin 1 and SNAP-25 (synaptosome-associated protein of 25 kDa) are t-SNAREs associated with the presynaptic membrane. The specific interaction of VAMPs/synaptobrevins with the syntaxin 1-SNAP-25 complex plays a fundamental role in the docking/fusion of synaptic vesicles with the presynaptic membrane (9-11).
Because of the central role of SNAREs in diverse vesicular transport steps, molecular identification, biochemical characterization, and subcellular localization of novel SNAREs constitute fundamentally important aspects of study in the field of vesicular transport. The Golgi apparatus plays a major role in the secretory pathway (1-4, 12). Currently, five distinct SNAREs have been shown to be associated with the Golgi apparatus in mammalian cells. These include syntaxin 5 (13-15), GS15 (16), GS27 (also termed membrin) (17-18), GS28 (also named GOS-28) (19-20), and syntaxin 6 (21-22). Syntaxin 5 and GS28 have both been shown to be involved in the endoplasmic reticulum (ER) to Golgi transport. GS28 has also been implicated in transport from the cis- to the medial-Golgi (15, 19-20). GS27 was shown to be involved in transport from the cis/medial- to trans-Golgi/trans Golgi network (18). The functional aspects of GS15 and syntaxin 6 remain to be established (16, 21-22). In this report, we describe the molecular, biochemical, and cell biological characterizations of Vti1-rp2, a novel 29-kDa SNARE associated with the Golgi apparatus. Vti1-rp2 is structurally homologous to Vti1p, a recently described yeast Golgi SNARE (23). We further show that Vti1-rp2 exists in distinct syntaxin 5- and syntaxin 6-containing SNARE complexes and is functionally important for protein trafficking in the secretory pathway.
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EXPERIMENTAL PROCEDURES |
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Materials-- Mouse EST clones (accession numbers AA016379 and W13616) were generated by the Washington University-Merck expressed sequence tag (EST) project and made available by IMAGE consortium via Research Genetics Inc. (Huntsville, Alabama). The mouse mRNA multiple tissues Northern blot was obtained from CLONTECH (Palo Alto, CA. Mouse monoclonal antibody against Golgi mannosidase II was from Babco (Berkeley, CA). Fluorescein isothiocyanate-conjugated goat anti-mouse IgG and rhodamine-conjugated goat anti-rabbit IgG were purchased from Boehringer Mannheim. Brefeldin A was from Epicentre Technologies.
cDNA Cloning and Sequencing-- Mouse EST clones were fully sequenced by the dideoxy chain termination method using a kit from U. S. Biochemical Corp. The complete coding region was assembled using the DNA Strider 1 program.
Northern Blot Analysis-- A mouse multiple tissue blot of poly(A)+ mRNA was probed with the insert of the EST clone AA016379 followed by actin probe as described previously (24).
Expression of Recombinant Proteins in Bacteria--
GST fusion
proteins were produced using the pGEX-KG vector (25) and purified as
described (24-25). Oligonucleotide 1 (5'- gctctagagtcttccgacttcgaagggtacgagcagg) and oligonucleotide 2 (5' ccgggtcgactcacagcatccccgtcagaatccgagagctcttccc) were used to retrieve the coding region for residues 1-185 by polymerase chain reaction using the EST clone AA016379 as the template. The polymerase chain
reaction product was digested with the restriction enzymes XbaI and SalI and then inserted into a similarly
digested pGEX-KG vector. After transformation of the Escherichia
coli strain DH5, colonies were screened for the production of
GST-Vti1-rp2 fusion protein. GST-
-SNAP has been described previously
and was purified accordingly (16). GST-Vti1-rp1 (31) was produced
similarly, and the details will be described elsewhere.
Preparation of Polyclonal Antibodies--
Rabbits were immunized
with GST-Vti1-rp2, and the specific antibodies were affinity-purified
as described previously (16, 24). Affinity-purified rabbit polyclonal
antibodies against syntaxin 5, syntaxin 6, syntaxin 7, and -COP have
been described previously (26-29).
Immunofluorescence Microscopy-- Immunofluorescence microscopy was performed as described previously (16, 24, 30).
Differential Extraction of Golgi Membranes and Immunoblot Analysis-- These were performed as described previously (16, 30).
In Vitro GST--SNAP Binding Assay and
Immunoprecipitation--
This was performed as described previously
(16, 26).
Micoinjection and in Vivo Transport of Vesicular Stomatitis Virus G-protein-- Vero cells grown on coverslips were infected with the vesicular stomatitis virus ts045 strain at 31 °C for 45 min and then shifted to 40 °C for 1 h. Cells were then transferred to 4 °C in Dulbecco's modified Eagle's medium without fetal bovine serum, and microinjection was performed under a Zeiss Axiophot microscopy using an Eppendorf micromanipulation system. Cells were transferred back to 40 °C immediately after injection and incubated at 40 °C for 2 h to accumulate the G-protein in the ER. Transport of G-protein was performed by incubating cells at 31 °C for 45 min in the presence of cycloheximide (to prevent new synthesis of G-protein). Cells were then fixed and processed for indirect immunofluorescence double-labeling to detect microinjected antibodies and the G-protein.
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RESULTS |
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Vti1-rp2, a Mammalian Protein Homologous to Yeast Vti1p-- Searching the EST data bases using the amino acid sequence of a novel Golgi SNARE characterized in the lab2 led to the identification of mouse EST clones (accession numbers AA016379 and W13616) that encode a putative SNARE. The EST clone W13616 was fully sequenced, and the nucleotide and the deduced amino acid sequences are shown in Fig. 1A. This protein was independently identified in three other laboratories and has been referred to as Vti1-rp2 (31), Vti1b (32), and Vti1a (33), respectively. To avoid further confusion in nomenclature, we have adopted the name Vti1-rp2 for this protein. Vti1-rp2 is a protein of 217 residues. Although the predicted molecular weight of Vti1-rp2 is 24,971 daltons, its apparent size as revealed by SDS-polyacrylamide gel electrophoresis is 29 kDa (see below). There exists a 22-residue carboxyl-terminal hydrophobic region that may function as a membrane anchor, a characteristic of the majority of known SNAREs (6-11, 16). Preceding the carboxyl-terminal hydrophobic tail are four regions (residues 32-62, 69-93, 112-134, 146-179) that have the potential to form coiled-coil structures as predicted by the COILS 2.1 program (Fig. 1B). A search of motifs with the ScanProsite program revealed the existence of an ATP/GTP binding motif A (P-loop) in the sequence (residues 170-177, ADANLGKS). Vti1-rp2 is homologous to Vti1p, a yeast Golgi SNARE implicated in at least two trafficking events (23). The homology between Vti1-rp2 and Vti1p occurs throughout the entire polypeptide with an overall amino acid identity of 28% and similarity of 45% (Fig. 1C). Since Vti1p does not contain the consensus ATP/GTP binding site motif A, it is not clear if the ATP/GTP binding site motif A observed in Vti1-rp2 is functionally important.
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Vti1-rp2 mRNA Is Widely Expressed-- To examine whether Vti1-rp2 is involved in a general cellular process or its function is restricted to certain tissues, Northern blot analysis was performed to examine the levels of Vti1-rp2 mRNA in various mouse tissues (Fig. 2A). A major mRNA species of about 2.6 kilobases was detected at varying levels in all the tissues examined, consistent with the notion that Vti1-rp2 may participate in an event common to all the cell types. Interestingly, the Vti1-rp2 mRNA detected in the testis has a smaller size (about 1.5 kilobases). The basis and significance for this different mRNA size of Vti1-rp2 in the testis is currently unknown.
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Vti1-rp2 Is a 29-kDa Integral Membrane Protein Associated with the
Golgi Apparatus--
The cytoplasmic domain (residues 1-185) of
Vti1-rp2 was expressed as a recombinant fusion protein with GST
(GST-Vti1-rp2). The purified GST-Vti1-rp2 was used to raise polyclonal
antibodies against Vti1-rp2 in rabbits. Although the predicted size for
Vti1-rp2 is 249710, its apparent size revealed by SDS-polyacrylamide
gel electrophoresis is about 29 kDa (Fig.
3A), because it migrates (lane 1) in between the 30-kDa marker (the marker lane) and
GS28 (lane 2) (a Golgi SNARE with an apparent size of 28 kDa) (20, 30). Another mammalian protein homologous to Vti1p has also been identified and has been referred to as Vtil-rp1 (31), Vti1 (32),
and Vti1b (33), respectively. To avoid further confusion, we have
adopted the name Vti1-rp1 for the other mammalian homolog of yeast
Vti1p. Since Vti1-rp2 displays significant amino acid sequence identity
(about 30%) with Vti1-rp1, it is essential to establish that our
affinity-purified antibodies do not cross-react with Vti1-rp1. As shown
(Fig. 3A), the detection of the 29-kDa protein in immunoblot
was selectively abolished by preincubation of antibodies with
recombinant cytoplasmic domain of Vti1-rp2 (lane 4) but not
with the cytoplasmic domain of Vti1-rp1 (lane 3),
establishing that our antibodies are specific for Vti1-rp2. Immunoblot
analysis revealed that Vti1-rp2 is enriched in Golgi membranes (Fig.
3B, lanes 4-6) as compared with total membranes, and microsomal membranes. The enrichment of Vti1-rp2 in Golgi membranes
is comparable with that of Golgi 2,6-sialyltransferase (lanes
1-3). When Golgi-enriched membranes were subjected to different extraction conditions (Fig. 3C), Vti1-rp2 was effectively
extracted by detergents but not by phosphate-buffered saline, 2.5 M urea, 0.1 M sodium bicarbonate (pH 12), or 2 M KCl. Vti1-rp2 is thus an integral membrane protein
enriched in the Golgi fractions.
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Vti1-rp2 Is a Novel Golgi SNARE--
To investigate whether
Vti1-rp2 indeed functions as a novel SNARE of the Golgi apparatus, we
examined the potential interaction of Vti1-rp2 with -SNAP. As shown
in Fig. 5A (upper
panel), Vti1-rp2 in the Golgi detergent extract was specifically
retained by immobilized GST-
-SNAP in a dose-dependent
manner. Under identical conditions, Vti1-rp2 was not retained by
immobilized GST or several other control GST fusion proteins (data not
shown). Furthermore, other Golgi proteins, including
2,6-sialyltransferase, were not retained by immobilized GST-
-SNAP
(Fig. 5A, lower panel). The interaction of
Vti1-rp2 with
-SNAP was further investigated (Fig. 5B).
Proteins in the Golgi extract were incubated with GST (lanes
1 and 4), GST-
-SNAP (lanes 2 and
5), and GST-
-SNAP (lanes 3 and 6);
after extensive washing, the beads (lanes 1-3) and 1/10 of
the supernatants (lane 4-6) were analyzed by immunoblot to
detect Vti1-rp2 (upper row) as well as GS28 (lower
row), which serves as a positive control. Vti1-rp2 was retained by
GST-
-SNAP as efficiently as GS28 (lanes 2 and
5). Neither Vti1-rp2 nor GS28 was retained by GST
(lanes 1 and 4). To lesser extents, Vti1-rp2 and
GS28 was significantly retained by GST-
-SNAP. These results
establish that interaction of Vti1-rp2 with GST-
-SNAP is specific
and occurs with efficiencies comparable with that of known Golgi SNAREs
such as GS28. Furthermore, interaction of Vti1-rp2 with immobilized
GST-
-SNAP could be abolished by NSF in conditions that promote
dissociation of SNARE complexes (Fig. 5C). When Golgi
extract was incubated with immobilized GST-
-SNAP in the presence of
increasing amounts of NSF in conditions (assembly buffer) that promote
formation of SNARE complexes (lanes 1-6), comparable
amounts of Vti1-rp2 were retained. However, retention of Vti1-rp2 by
immobilized GST-
-SNAP was readily abolished by NSF in conditions
(lane 7-12) that promote ATP hydrolysis by NSF and
disassembly of SNARE complexes. These results not only further confirmed that the interaction of Vti1-rp2 with
-SNAP is specific but also revealed that the interaction of Vti1-rp2 with
-SNAP is in
the context of Vti1-rp2-containing SNARE complexes.
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Coimmunoprecipitation of Vti1-rp2 with Syntaxin 5 and 6-- To gain additional understanding of Vti1-rp2 as a Golgi SNARE, we investigated the potential interaction of Vti1-rp2 with two Golgi t-SNAREs, the cis-Golgi syntaxin 5 (13-15, 17) and the trans-Golgi network syntaxin 6 (21-22). Golgi detergent extracts were immunoprecipitated with antibodies against syntaxin 5, syntaxin 6, or control rabbit IgG. The immunoprecipitates (beads) (lanes 1, 2, 5, and 6) and 1/10 of the supernatants (lanes 3, 4, 7, and 8) were analyzed by immunoblot analysis. As shown in Fig. 6 (lanes 1-4), syntaxin 5 was efficiently precipitated by its antibodies (lanes 1 and 3) but not by control antibodies (lanes 2 and 4). Vti1-rp2 was clearly coimmunoprecipitated by antibodies against syntaxin 5 (lanes 1 and 3) but not by control antibodies (lanes 2 and 4), suggesting that a significant fraction of Vti1-rp2 exists in a syntaxin 5-containing SNARE complex. Usually, about 3-5% of Vti1-rp2 could be coimmunoprecipitated by antibodies against syntaxin 5. Under identical conditions, syntaxin 6 was not coimmunoprecipitated by antibodies against syntaxin 5 (lanes 1 and 3). When Golgi extracts were immunoprecipitated with antibodies against syntaxin 6 (lanes 5-8), essentially all syntaxin 6 was precipitated (lanes 5 and 7). Furthermore, Vti1-rp2 was obviously coimmunoprecipitated by anti-syntaxin 6 antibodies (lanes 5 and 7) but not by control control IgG (lanes 6 and 8). About 10-15% of Vti1-rp2 is routinely coimmunoprecipitated by antibodies against syntaxin 6. In contrast, syntaxin 5 was not coimmunoprecipitated by anti-syntaxin 6 antibodies (lanes 5 and 7). These results suggest that significant amounts of Vti1-rp2 exist in at least two distinct SNARE complexes, one containing syntaxin 5 and the other containing syntaxin 6. This conclusion was further substantiated by our observation that significant amounts of syntaxin 5 and syntaxin 6 were coimmunoprecipitated by antibodies against Vti1-rp2 (data not shown).
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A Role of Vti1-rp2 in Protein Transport in the Secretory
Pathway--
The association of Vti1-rp2 with the Golgi apparatus and
its establishment as a SNARE suggest that it may participate in protein trafficking in the secretory pathway. To investigate this, Vero cells
grown on coverslips were first infected with vesicular stomatitis virus
ts045 and then microinjected with affinity-purified antibodies against
Vti1-rp2. Transport of G-protein along the secretory pathway was
monitored by indirect immunofluorescence microscopy. Since microinjection of antibodies against EAGE epitope of -COP was shown
previously to inhibit G-protein transport (29), cells microinjected
with
-COP antibodies serve as the positive control. We have shown
recently that syntaxin 7 is in the endosomal compartment (28), and
syntaxin 7 is thus not expected to function in the secretory pathway.
Cells microinjected with syntaxin 7 antibodies thus serve as a negative
control. As shown in Fig. 7, in cells microinjected with antibodies against Vti1-rp2 (C,
arrows), surface labeling of G-protein was dramatically
reduced, resulting in accumulation of G-protein in perinuclear
structures characteristic of the Golgi apparatus (D,
arrows). This inhibitory effect is comparable with that seen
in cells microinjected with antibodies against
-COP (A-B, arrows). In marked contrast, transport of
G-protein to the cell surface was unaffected (E and
F, arrows) in cells microinjected with syntaxin 7 antibodies. These results suggest that transport of G-protein from the
ER to the plasma membrane is specifically inhibited in cells
microinjected with antibodies against Vti1-rp2, and the site of
inhibition seems to be at the level of the Golgi apparatus because
G-protein was seen to accumulate in structures characteristic of the
Golgi apparatus and the arrested G-protein colocalized well with
markers of the Golgi apparatus such as
12-(N-methyl-N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl))-ceramide and binding sites for lectin lens culinaris agglutinin (data not shown). Although more detailed future experiments are needed to address
the mechanistic aspects of Vti1-rp2 involvement in protein transport,
these results clearly revealed a role of Vti1-rp2 in protein transport
in the secretory pathway.
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DISCUSSION |
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We have identified a novel 29-kDa mammalian protein (Vti1-rp2)
that has characteristics of a SNARE based on the presence of a
COOH-terminal hydrophobic membrane anchor and several regions that can
potentially form coiled-coil structures (6-11, 16). Three observations
establish that Vti1-rp2 is indeed a SNARE. First, Vti1-rp2 in Golgi
detergent extract can interact with immobilized GST--SNAP in a
specific and dose-dependent manner. Interaction of Vti1-rp2
with immobilized GST-
-SNAP occurs with efficiencies comparable with
that of known Golgi SNAREs such as GS28 (20). The second line of
evidence is that association of Vti1-rp2 with GST-
-SNAP could be
abolished by NSF, specifically under conditions that promote NSF ATPase
activity and dissociation of SNARE complexes. This suggests that
interaction of Vti1-rp2 with immobilized
-SNAP occurs through
Vti1-rp2-containing SNARE complex(es) in the Golgi extract. The
demonstration of existence of Vti1-rp2 in at least two distinct SNARE
complexes (one containing syntaxin 5 and the other containing syntaxin
6) provides the third line of evidence to support that Vti1-rp2 is a
novel SNARE. The subcellular localization of Vti1-rp2 was established
by two independent results. First, Vti1-rp2 is highly enriched in a
membrane fraction that is also enriched for the Golgi apparatus.
Furthermore, Vti1-rp2 colocalized well with the Golgi marker
mannosidase II in both control as well as nocodazole-fragmented Golgi
apparatus. Like mannosidase II, Vti1-rp2 could be redistributed into
ER-like structures by brefeldin A. It is thus firmly established that
Vti1-rp2 is a novel SNARE of the Golgi apparatus.
Data base searches with Vti1-rp2 sequence revealed that Vti1-rp2 is most homologous to Vti1p. Vti1p is a recently identified v-SNARE of the yeast Golgi and has been implicated in two independent vesicular transport events (23). By interacting with the early Golgi t-SNARE Sed5p (the yeast counterpart of syntaxin 5) (37-38), Vti1p has been suggested to function as a v-SNARE for vesicles involved in retrograde intra-Golgi transport. Furthermore, Vti1p has also been shown to be involved in transport from the late Golgi to the vacuole (equivalent to the mammalian lysosome) by interacting with Pep12p (39), a syntaxin-like t-SNARE of the pre-vacuolar compartment (equivalent to the mammalian late endosome). Whether Vti1-rp2 represents the mammalian counterpart of Vti1p remains to be further investigated, although another mammalian protein (Vti1-rp1) homologous to Vti1p could functionally substitute for the yeast Vti1p (32). Besides its sequence homology with Vti1p, another property of Vti1-rp2 that is similar to Vti1p is that a significant amount of Vti1-rp2 exists in a syntaxin 5-containing Golgi SNARE complex. In addition, a significant amount of Vti1-rp2 was also shown to be present in a syntaxin 6-containing SNARE complex. Since coimmunoprecipitation of syntaxin 5 and syntaxin 6 was not observed, these results suggest that Vti1-rp2 exists in distinct syntaxin 5- and syntaxin 6-containing SNARE complexes. Although the functional aspects remain to be established, syntaxin 6 has been shown recently to be enriched in the trans-Golgi network (22).
The presence of Vti1-rp2 in at least two distinct Golgi SNARE complexes
indicates that it may function as a SNARE for at least two types of
vesicle-mediated transport events. One will dock and fuse with
cis-Golgi by interaction with syntaxin 5, whereas the other will dock
and fuse with trans-Golgi network via interaction with syntaxin 6. The
interaction of Vti1-rp2 with at least two syntaxin-like t-SNAREs is
consistent with a recent study showing that yeast Vti1p could interact
with at least five distinct syntaxin-like t-SNAREs (40). Since Vti1p
participate in two distinct transport events (one associated with the
secretory pathway and the other in the endosomal pathway), the
existence of two distinct mammalian proteins homologous to Vti1p
indicates that the two equivalent transport events in mammalian cells
may be mediated by two distinct proteins. The preferential association
of Vti1-rp2 with the Golgi apparatus indicates that Vti1-rp2 may
participate in a transport event in the secretory pathway. Consistent
with this, we have shown that microinjection of antibodies against
Vti1-rp2 specifically inhibited transport of G-protein to the cell
surface at the level of Golgi apparatus. The extents of inhibition of
G-protein transport seen in cells microinjected with Vti1-rp2
antibodies are comparable with those seen in cells microinjected with
antibodies against -coat protein. Serving as a negative control,
G-protein transport to the plasma membrane was unaffected in cells
microinjected with antibodies against endosomal syntaxin 7. Vti1-rp2
thus plays a role in protein transport in the secretory pathway, and
the role of yeast Vti1p in the secretory pathway is most likely
mediated by Vti1-rp2 in mammalian cells. Furthermore, our preliminary
studies with Vti1-rp1 suggests that it is preferentially associated
with the trans-Golgi network and/or the endosomal
compartment,3 indicating that
the endosomal role of yeast Vti1p is most likely mediated by Vti1-rp1
in mammalian cells.
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ACKNOWLEDGEMENTS |
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We thank James E. Rothman for his generous
gift of plasmids for producing recombinant HisX6-NSF and
HisX6--SNAP, Richard Scheller for rat syntaxin 5 and syntaxin 6 cDNA clones, members of the Hong laboratory for critical reading of
the manuscript, and Y. H. Tan for his continuous support.
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FOOTNOTES |
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* This work was funded by the Institute of Molecular and Cell Biology, National University of Singapore (to W. H.).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.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF035823.
To whom correspondence should be addressed: Institute of Molecular
and Cell Biology, 30 Medical Dr., Singapore 117609, Singapore. Tel.:
65-778-6827; Fax: 65-779-1117; E-mail: mcbhwj{at}imcb.nus.edu.sg.
The abbreviations used are: NSF, N-ethylmaleimide-sensitive factorSNAP, soluble NSF attachment proteinsSNARE, SNAP receptorv-SNARE, vesicle-associated SNAREt-SNARE, target membrane SNAREER, endoplasmic reticulumEST, expressed sequence tagGST, glutathione S- transferase.
2 S. H. Wong and W. H. Hong, manuscript in preparation.
3 S. H. Wong and W.-J. Hong, unpublished observations.
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REFERENCES |
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