A 29-Kilodalton Golgi SolubleN-Ethylmaleimide-sensitive Factor Attachment Protein Receptor (Vti1-rp2) Implicated in Protein Trafficking in the Secretory Pathway
1998; Elsevier BV; Volume: 273; Issue: 34 Linguagem: Inglês
10.1074/jbc.273.34.21783
ISSN1083-351X
AutoresYue Xu, Siew Heng Wong, Bor Luen Tang, V. Nathan Subramaniam, Tao Zhang, Wanjin Hong,
Tópico(s)Biochemical and Structural Characterization
ResumoExpressed 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 α soluble N-ethylmaleimide-sensitive factor attachment protein (GST-α-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. 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 α soluble N-ethylmaleimide-sensitive factor attachment protein (GST-α-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. Participation of NSF 1The abbreviations used are: NSFN-ethylmaleimide-sensitive factorSNAPsoluble NSF attachment proteinsSNARESNAP receptorv-SNAREvesicle-associated SNAREt-SNAREtarget membrane SNAREERendoplasmic reticulumESTexpressed sequence tagGSTglutathione S- transferase. 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) (1Pryer N.K. Wuestehube L.J. Schekman R. Annu. Rev. Biochem. 1992; 61: 471-516Crossref PubMed Scopus (369) Google Scholar, 2Rothman J.E. Nature. 1994; 372: 55-63Crossref PubMed Scopus (2011) Google Scholar, 3Rothman J.E. Wieland F.T. Science. 1996; 272: 227-234Crossref PubMed Scopus (1026) Google Scholar, 4Schekman R. Orci L. Science. 1996; 271: 1526-1532Crossref PubMed Scopus (819) Google Scholar, 5Whiteheart S.W. Kubalek E.W. Trends Cell Biol. 1995; 5: 64-69Abstract Full Text PDF PubMed Scopus (80) Google Scholar). 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) (6Ferro-Novick S. Jahn R. Nature. 1994; 370: 191-193Crossref PubMed Scopus (561) Google Scholar, 7Pfeffer S.R. Annu. Rev. Cell Dev. Biol. 1996; 12: 441-461Crossref PubMed Scopus (180) Google Scholar, 8Rothman J.E. Warren G. Curr. Biol. 1994; 4: 220-233Abstract Full Text Full Text PDF PubMed Scopus (481) Google Scholar, 9Scheller R.H. Neuron. 1995; 14: 893-897Abstract Full Text PDF PubMed Scopus (196) Google Scholar, 10Söllner T. Whiteheart S.W. Brunner M. Erdjument-Bromage H. Geromanos S. Tempst P. Rothman J.E. Nature. 1993; 362: 318-324Crossref PubMed Scopus (2637) Google Scholar, 11Südhof T.C. Nature. 1995; 375: 645-653Crossref PubMed Scopus (1770) Google Scholar). This v-/t-SNARE pairing is a key event in the docking and fusion of the vesicle with its specific target membrane (6Ferro-Novick S. Jahn R. Nature. 1994; 370: 191-193Crossref PubMed Scopus (561) Google Scholar, 7Pfeffer S.R. Annu. Rev. Cell Dev. Biol. 1996; 12: 441-461Crossref PubMed Scopus (180) Google Scholar, 8Rothman J.E. Warren G. Curr. Biol. 1994; 4: 220-233Abstract Full Text Full Text PDF PubMed Scopus (481) Google Scholar, 9Scheller R.H. Neuron. 1995; 14: 893-897Abstract Full Text PDF PubMed Scopus (196) Google Scholar, 10Söllner T. Whiteheart S.W. Brunner M. Erdjument-Bromage H. Geromanos S. Tempst P. Rothman J.E. Nature. 1993; 362: 318-324Crossref PubMed Scopus (2637) Google Scholar, 11Südhof T.C. Nature. 1995; 375: 645-653Crossref PubMed Scopus (1770) Google Scholar). 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 (9Scheller R.H. Neuron. 1995; 14: 893-897Abstract Full Text PDF PubMed Scopus (196) Google Scholar, 10Söllner T. Whiteheart S.W. Brunner M. Erdjument-Bromage H. Geromanos S. Tempst P. Rothman J.E. Nature. 1993; 362: 318-324Crossref PubMed Scopus (2637) Google Scholar, 11Südhof T.C. Nature. 1995; 375: 645-653Crossref PubMed Scopus (1770) Google Scholar). N-ethylmaleimide-sensitive factor soluble NSF attachment proteins SNAP receptor vesicle-associated SNARE target membrane SNARE endoplasmic reticulum expressed sequence tag glutathione S- transferase. 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 (1Pryer N.K. Wuestehube L.J. Schekman R. Annu. Rev. Biochem. 1992; 61: 471-516Crossref PubMed Scopus (369) Google Scholar, 2Rothman J.E. Nature. 1994; 372: 55-63Crossref PubMed Scopus (2011) Google Scholar, 3Rothman J.E. Wieland F.T. Science. 1996; 272: 227-234Crossref PubMed Scopus (1026) Google Scholar, 4Schekman R. Orci L. Science. 1996; 271: 1526-1532Crossref PubMed Scopus (819) Google Scholar, 12Palade G.E. Science. 1975; 189: 347-354Crossref PubMed Scopus (2354) Google Scholar). Currently, five distinct SNAREs have been shown to be associated with the Golgi apparatus in mammalian cells. These include syntaxin 5 (13Banfield D.K. Lewis M.J. Rabouille C. Warren G. Pelham H.R.B. J. Cell Biol. 1994; 127: 357-371Crossref PubMed Scopus (147) Google Scholar, 14Bennett M.K. Garcia-Arraras J.E. Elferink L.A. Peterson K. Fleming A.M. Hazuka C.D. Scheller R.H. Cell. 1993; 74: 863-873Abstract Full Text PDF PubMed Scopus (591) Google Scholar, 15Dascher C. Matteson J. Balch W.E. J. Biol. Chem. 1994; 269: 29363-29366Abstract Full Text PDF PubMed Google Scholar), GS15 (16Xu Y. Wong S.H. Zhang T. Subramaniam V.N. Hong W. J. Biol. Chem. 1997; 272: 20162-20166Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar), GS27 (also termed membrin) (17Hay J.C. Chao D.S. Kuo C.S. Scheller R.H. Cell. 1997; 89: 147-158Abstract Full Text Full Text PDF Scopus (177) Google Scholar, 18Lowe S.L. Peter F. Subramaniam V.N. Wong S.H. Hong W. Nature. 1997; 389: 881-883Crossref PubMed Scopus (69) Google Scholar), GS28 (also named GOS-28) (19Nagahama M. Orci L. Ravazzola M. Amherdt M. Lacomis L. Tempst P. Rothman J.E. Söllner T.H. J. Cell Biol. 1996; 133: 507-516Crossref PubMed Scopus (94) Google Scholar, 20Subramaniam V.N. Peter F. Philip R. Wong S.H. Hong W. Science. 1996; 272: 1161-1163Crossref PubMed Scopus (122) Google Scholar), and syntaxin 6 (21Bock J.B. Lin R.C. Scheller R.H. J. Biol. Chem. 1996; 271: 17961-17965Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar, 22Bock J.B. Klumperman J. Davanger S. Scheller R.H. Mol. Biol. Cell. 1997; 8: 1261-1271Crossref PubMed Scopus (248) Google Scholar). 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 (15Dascher C. Matteson J. Balch W.E. J. Biol. Chem. 1994; 269: 29363-29366Abstract Full Text PDF PubMed Google Scholar, 19Nagahama M. Orci L. Ravazzola M. Amherdt M. Lacomis L. Tempst P. Rothman J.E. Söllner T.H. J. Cell Biol. 1996; 133: 507-516Crossref PubMed Scopus (94) Google Scholar, 20Subramaniam V.N. Peter F. Philip R. Wong S.H. Hong W. Science. 1996; 272: 1161-1163Crossref PubMed Scopus (122) Google Scholar). GS27 was shown to be involved in transport from the cis/medial- to trans-Golgi/trans Golgi network (18Lowe S.L. Peter F. Subramaniam V.N. Wong S.H. Hong W. Nature. 1997; 389: 881-883Crossref PubMed Scopus (69) Google Scholar). The functional aspects of GS15 and syntaxin 6 remain to be established (16Xu Y. Wong S.H. Zhang T. Subramaniam V.N. Hong W. J. Biol. Chem. 1997; 272: 20162-20166Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar, 21Bock J.B. Lin R.C. Scheller R.H. J. Biol. Chem. 1996; 271: 17961-17965Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar, 22Bock J.B. Klumperman J. Davanger S. Scheller R.H. Mol. Biol. Cell. 1997; 8: 1261-1271Crossref PubMed Scopus (248) Google Scholar). 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 (23von Mollard G.F. Nothwehr S.F. Stevens T.H. J. Cell Biol. 1997; 137: 1511-1524Crossref PubMed Scopus (175) Google Scholar). 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. Mouse EST clones (accession numbers AA016379 andW13616) 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 fromCLONTECH (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. 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. 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 (24Lowe S.L. Wong S.H. Hong W. J. Cell Sci. 1996; 109: 209-220Crossref PubMed Google Scholar). GST fusion proteins were produced using the pGEX-KG vector (25Guan K. Dixon J.E. Anal. Biochem. 1991; 192: 262-267Crossref PubMed Scopus (1641) Google Scholar) and purified as described (24Lowe S.L. Wong S.H. Hong W. J. Cell Sci. 1996; 109: 209-220Crossref PubMed Google Scholar, 25Guan K. Dixon J.E. Anal. Biochem. 1991; 192: 262-267Crossref PubMed Scopus (1641) Google Scholar). 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 enzymesXbaI and SalI and then inserted into a similarly digested pGEX-KG vector. After transformation of the Escherichia coli strain DH5α, colonies were screened for the production of GST-Vti1-rp2 fusion protein. GST-α-SNAP has been described previously and was purified accordingly (16Xu Y. Wong S.H. Zhang T. Subramaniam V.N. Hong W. J. Biol. Chem. 1997; 272: 20162-20166Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar). GST-Vti1-rp1 (31Lupashin V.V. Pokrovskaya I.D. McNew J.A. Waters M.G. Mol. Biol. Cell. 1997; 8: 2659-2676Crossref PubMed Scopus (92) Google Scholar) was produced similarly, and the details will be described elsewhere. Rabbits were immunized with GST-Vti1-rp2, and the specific antibodies were affinity-purified as described previously (16Xu Y. Wong S.H. Zhang T. Subramaniam V.N. Hong W. J. Biol. Chem. 1997; 272: 20162-20166Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar, 24Lowe S.L. Wong S.H. Hong W. J. Cell Sci. 1996; 109: 209-220Crossref PubMed Google Scholar). Affinity-purified rabbit polyclonal antibodies against syntaxin 5, syntaxin 6, syntaxin 7, and β-COP have been described previously (26Subramaniam V.N. Loh E. Hong W. J. Biol. Chem. 1997; 272: 25441-25444Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar, 27Zhang T. Wong S.H. Tang B.L. Xu Y. Peter F. Hong W. J. Cell Biol. 1997; 139: 1157-1168Crossref PubMed Scopus (55) Google Scholar, 28Wong S.H. Xu Y. Zhang T. Hong W. J. Biol. Chem. 1998; 273: 375-380Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar, 29Pepperkok R. Scheel J. Horstmann H. Hauri H.P. Griffiths G. Kreis T.E. Cell. 1993; 74: 71-82Abstract Full Text PDF PubMed Scopus (275) Google Scholar). Immunofluorescence microscopy was performed as described previously (16Xu Y. Wong S.H. Zhang T. Subramaniam V.N. Hong W. J. Biol. Chem. 1997; 272: 20162-20166Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar, 24Lowe S.L. Wong S.H. Hong W. J. Cell Sci. 1996; 109: 209-220Crossref PubMed Google Scholar, 30Subramaniam V.N. Krijnse-Locker J. Tang B.L. Ericsson M. Yusoff A.R.B.M. Griffiths G. Hong W. J. Cell Sci. 1995; 108: 2405-2414PubMed Google Scholar). These were performed as described previously (16Xu Y. Wong S.H. Zhang T. Subramaniam V.N. Hong W. J. Biol. Chem. 1997; 272: 20162-20166Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar,30Subramaniam V.N. Krijnse-Locker J. Tang B.L. Ericsson M. Yusoff A.R.B.M. Griffiths G. Hong W. J. Cell Sci. 1995; 108: 2405-2414PubMed Google Scholar). This was performed as described previously (16Xu Y. Wong S.H. Zhang T. Subramaniam V.N. Hong W. J. Biol. Chem. 1997; 272: 20162-20166Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar, 26Subramaniam V.N. Loh E. Hong W. J. Biol. Chem. 1997; 272: 25441-25444Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar). 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. Searching the EST data bases using the amino acid sequence of a novel Golgi SNARE characterized in the lab 2S. H. Wong and W. H. Hong, manuscript in preparation. led to the identification of mouse EST clones (accession numbers AA016379 andW13616) 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. 1 A. This protein was independently identified in three other laboratories and has been referred to as Vti1-rp2 (31Lupashin V.V. Pokrovskaya I.D. McNew J.A. Waters M.G. Mol. Biol. Cell. 1997; 8: 2659-2676Crossref PubMed Scopus (92) Google Scholar), Vti1b (32von Mollard G.F. Stevens T.H. J. Biol. Chem. 1998; 273: 2624-2630Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar), and Vti1a (33Advani R.J. Bae H.-R. Bock J.B. Chao D.S. Doung Y.-C. Prekeris R. Yoo J.-S. Scheller R.H. J. Biol. Chem. 1998; 273: 10317-10324Abstract Full Text Full Text PDF PubMed Scopus (223) Google Scholar), 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 (6Ferro-Novick S. Jahn R. Nature. 1994; 370: 191-193Crossref PubMed Scopus (561) Google Scholar, 7Pfeffer S.R. Annu. Rev. Cell Dev. Biol. 1996; 12: 441-461Crossref PubMed Scopus (180) Google Scholar, 8Rothman J.E. Warren G. Curr. Biol. 1994; 4: 220-233Abstract Full Text Full Text PDF PubMed Scopus (481) Google Scholar, 9Scheller R.H. Neuron. 1995; 14: 893-897Abstract Full Text PDF PubMed Scopus (196) Google Scholar, 10Söllner T. Whiteheart S.W. Brunner M. Erdjument-Bromage H. Geromanos S. Tempst P. Rothman J.E. Nature. 1993; 362: 318-324Crossref PubMed Scopus (2637) Google Scholar, 11Südhof T.C. Nature. 1995; 375: 645-653Crossref PubMed Scopus (1770) Google Scholar, 16Xu Y. Wong S.H. Zhang T. Subramaniam V.N. Hong W. J. Biol. Chem. 1997; 272: 20162-20166Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar). 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.1 B). 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 (23von Mollard G.F. Nothwehr S.F. Stevens T.H. J. Cell Biol. 1997; 137: 1511-1524Crossref PubMed Scopus (175) Google Scholar). 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. 1 C). 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. 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.2 A). 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. 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.3 A), 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) (20Subramaniam V.N. Peter F. Philip R. Wong S.H. Hong W. Science. 1996; 272: 1161-1163Crossref PubMed Scopus (122) Google Scholar, 30Subramaniam V.N. Krijnse-Locker J. Tang B.L. Ericsson M. Yusoff A.R.B.M. Griffiths G. Hong W. J. Cell Sci. 1995; 108: 2405-2414PubMed Google Scholar). Another mammalian protein homologous to Vti1p has also been identified and has been referred to as Vtil-rp1 (31Lupashin V.V. Pokrovskaya I.D. McNew J.A. Waters M.G. Mol. Biol. Cell. 1997; 8: 2659-2676Crossref PubMed Scopus (92) Google Scholar), Vti1 (32von Mollard G.F. Stevens T.H. J. Biol. Chem. 1998; 273: 2624-2630Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar), and Vti1b (33Advani R.J. Bae H.-R. Bock J.B. Chao D.S. Doung Y.-C. Prekeris R. Yoo J.-S. Scheller R.H. J. Biol. Chem. 1998; 273: 10317-10324Abstract Full Text Full Text PDF PubMed Scopus (223) Google Scholar), 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. 3 A), 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.3 B, 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 α2,6-sialyltransferase (lanes 1–3). When Golgi-enriched membranes were subjected to different extraction conditions (Fig. 3 C), Vti1-rp2 was effectively extracted by detergents but not by phosphate-buffered saline, 2.5m urea, 0.1 m sodium bicarbonate (pH 12), or 2m KCl. Vti1-rp2 is thus an integral membrane protein enriched in the Golgi fractions. Indirect immunofluorescence microscopy was used to examine the exact subcellular localization of Vti1-rp2 (Fig.4). Affinity-purified antibodies against Vti1-rp2 specifically labeled perinuclear structures (Fig.4 A, panel a) characteristic of the Golgi apparatus (34Louvard D. Reggio H. Warren G. J. Cell Biol. 1982; 92: 92-107Crossref PubMed Scopus (228) Google Scholar), and this labeling colocalized well with that of Golgi mannosidase II (panel b) (35Moreman K.W. Robbins P.W. J. Cell Biol. 1991; 115: 1521-1534Crossref PubMed Scopus (118) Google Scholar). When the Golgi apparatus was fragmented by nocodazole treatment (panels c–d), Vti1-rp2 and mannosidase II were colocalized well in the fragmented Golgi apparatus. Similar to mannosidase II and other Golgi proteins (36Klausner R.D. Donaldson J.G. Lippincott-Schwartz J. J. Cell Biol. 1992; 116: 1071-1080Crossref PubMed Scopus (1548) Google Scholar), Vti1-rp2 was redistributed to the ER when cells were treated with brefeldin A (e–f). These results firmly establish that Vti1-rp2 is an integral membrane protein associated preferentially with the Golgi apparatus. To investigate whether Vti1-rp2 indeed functions as a novel SNARE of the Golgi apparatus, we examined the potential interaction of Vti1-rp2 with α-SNAP. As shown in Fig. 5 A (upper panel), Vti1-rp2 in the Golgi detergent extract was specifically retained by immobilized GST-α-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 α2,6-sialyltransferase, were not retained by immobilized GST-α-SNAP (Fig. 5 A, lower panel). The interaction of Vti1-rp2 with α-SNAP was further investigated (Fig. 5 B). Proteins in the Golgi extract were incubated with GST (lanes 1 and 4), GST-α-SNAP (lanes 2 and5), and GST-γ-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-α-SNAP as efficiently as GS28 (lanes 2 and5). 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-γ-SNAP. These results establish that interaction of Vti1-rp2 with GST-α-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-α-SNAP could be abolished by NSF in conditions that promote dissociation of SNARE complexes (Fig. 5 C). When Golgi extract was incubated with immobilized GST-α-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-α-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 α-SNAP is specific but also revealed that the interaction of Vti1-rp2 with α-SNAP is in the context of Vti1-rp2-containing SNARE complexes. 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 (13Banfield D.K. Lewis M.J. Rabouille C. Warren G. Pelham H.R.B. J. Cell Biol. 1994; 127: 357-371Crossref PubMed Scopus (147) Google Scholar, 14Bennett M.K. Garcia-Arraras J.E. Elferink L.A. Peterson K. Fleming A.M. Hazuka C.D. Scheller R.H. Cell. 1993; 74: 863-873Abstract Full Text PDF PubMed Scopus (591) Google Scholar, 15Dascher C. Matteson J. Balch W.E. J. Biol. Chem. 1994; 269: 29363-29366Abstract Full Text PDF PubMed Google Scholar, 17Hay J.C. Chao D.S. Kuo C.S. Scheller R.H. Cell. 1997; 89: 147-158Abstract Full Text Full Text PDF Scopus (177) Google Scholar) and the trans-Golgi network syntaxin 6 (21Bock J.B. Lin R.C. Scheller R.H. J. Biol. Chem. 1996; 271: 17961-17965Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar, 22Bock J.B. Klumperman J. Davanger S. Scheller R.H. Mol. Biol. Cell. 1997; 8: 1261-1271Crossref PubMed Scopus (248) Google Scholar). 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). 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 β-COP was shown previously to inhibit G-protein transport (29Pepperkok R. Scheel J. Horstmann H. Hauri H.P. Griffiths G. Kreis T.E. Cell. 1993; 74: 71-82Abstract Full Text PDF PubMed Scopus (275) Google Scholar), cells microinjected with β-COP antibodies serve as the positive control. We have shown recently that syntaxin 7 is in the endosomal compartment (28Wong S.H. Xu Y. Zhang T. Hong W. J. Biol. Chem. 1998; 273: 375-380Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar), 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 β-COP (A–B, arrows). In marked contrast, transport of G-protein to the cell surface was unaffected (E andF, 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. 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 (6Ferro-Novick S. Jahn R. Nature. 1994; 370: 191-193Crossref PubMed Scopus (561) Google Scholar, 7Pfeffer S.R. Annu. Rev. Cell Dev. Biol. 1996; 12: 441-461Crossref PubMed Scopus (180) Google Scholar, 8Rothman J.E. Warren G. Curr. Biol. 1994; 4: 220-233Abstract Full Text Full Text PDF PubMed Scopus (481) Google Scholar, 9Scheller R.H. Neuron. 1995; 14: 893-897Abstract Full Text PDF PubMed Scopus (196) Google Scholar, 10Söllner T. Whiteheart S.W. Brunner M. Erdjument-Bromage H. Geromanos S. Tempst P. Rothman J.E. Nature. 1993; 362: 318-324Crossref PubMed Scopus (2637) Google Scholar, 11Südhof T.C. Nature. 1995; 375: 645-653Crossref PubMed Scopus (1770) Google Scholar, 16Xu Y. Wong S.H. Zhang T. Subramaniam V.N. Hong W. J. Biol. Chem. 1997; 272: 20162-20166Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar). Three observations establish that Vti1-rp2 is indeed a SNARE. First, Vti1-rp2 in Golgi detergent extract can interact with immobilized GST-α-SNAP in a specific and dose-dependent manner. Interaction of Vti1-rp2 with immobilized GST-α-SNAP occurs with efficiencies comparable with that of known Golgi SNAREs such as GS28 (20Subramaniam V.N. Peter F. Philip R. Wong S.H. Hong W. Science. 1996; 272: 1161-1163Crossref PubMed Scopus (122) Google Scholar). The second line of evidence is that association of Vti1-rp2 with GST-α-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 α-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 (23von Mollard G.F. Nothwehr S.F. Stevens T.H. J. Cell Biol. 1997; 137: 1511-1524Crossref PubMed Scopus (175) Google Scholar). By interacting with the early Golgi t-SNARE Sed5p (the yeast counterpart of syntaxin 5) (37Hardwick K.G. Pelham H.R.B. J. Cell Biol. 1992; 119: 513-521Crossref PubMed Scopus (256) Google Scholar, 38Sögaard M. Tani K. Ye R.B. Geromanos S. Tempst P. Kirchhausen T. Rothman J.E. Söllner T. Cell. 1994; 78: 937-948Abstract Full Text PDF PubMed Scopus (443) Google Scholar), 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 (39Becherer K.A. Rieder S.E. Emr S.D. Jones E.W. Mol. Biol. Cell. 1996; 7: 579-594Crossref PubMed Scopus (253) Google Scholar), 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 (32von Mollard G.F. Stevens T.H. J. Biol. Chem. 1998; 273: 2624-2630Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar). 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 (22Bock J.B. Klumperman J. Davanger S. Scheller R.H. Mol. Biol. Cell. 1997; 8: 1261-1271Crossref PubMed Scopus (248) Google Scholar). 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 (40Holthuis J.C.M. Nichols B.J. Dhruvakumar S. Pelham H.R.B. EMBO J. 1998; 17: 113-126Crossref PubMed Scopus (218) Google Scholar). 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 β-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, 3S. H. Wong and W.-J. Hong, unpublished observations. indicating that the endosomal role of yeast Vti1p is most likely mediated by Vti1-rp1 in mammalian cells. We thank James E. Rothman for his generous gift of plasmids for producing recombinant HisX6-NSF and HisX6-α-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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