- Email: [email protected]
Syntaxin 10: A Member of the Syntaxin Family Localized to theTrans-Golgi Network
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS ARTICLE NO.
242, 345–350 (1998)
RC977966
Syntaxin 10: A Member of the Syntaxin Family Localized to the Trans-Golgi Network Bor Luen Tang,1 Delphine Y. H. Low,1 Andrew E. H. Tan, and Wanjin Hong2 Membrane Biology Laboratory, Institute of Molecular and Cell Biology, 30 Medical Drive, Singapore 117609, Republic of Singapore
Received December 1, 1997
We have cloned a new member of the syntaxin family of proteins, designated human syntaxin 10 (hsyn10). The open reading frame encodes a polypeptide of 249 amino acids with potential coiled-coil domains and a carboxy-terminal hydrophobic tail. hsyn10 is particularly homologous to the recently reported rat syntaxin 6 (about 60% identity). Northern blot analysis showed that the transcript is enriched in the heart, skeletal muscles and pancreas. Indirect immunofluorescence studies using polyclonal antibodies raised against recombinant protein showed that the protein is localized to intracellular membrane structures, with perinuclear staining patterns colocalising well with the Golgi SNARE GS28. Morphological alterations of the staining pattern of the protein with brefeldin A but not wortmannin treatment indicate that the protein is localize to the transGolgi network. q 1998 Academic Press
Membranous compartments in eukaryotic cells are linked by vesicular transport processes (16, 17). Molecular components required for both vesicle budding (4, 26) and vesicle docking/fusion processes (16-20) have been isolated. The N-ethylmaleimide sensitive factor (NSF), an ATPase whose activity regulates the formation and dissociation of fusion complexes works in conjunction with another soluble factor, the soluble NSF attachment protein (SNAP), in mediating vesicle docking and/or fusion (17). SNAP receptors, or SNAREs, 1
The first two authors contributed equally. Correspondence. Fax: (65) 779-1117. E-mail: mcbhwj@leonis. nus.sg. Abbreviations used: ER, endoplasmic reticulum; FBS, fetal bovine serum; FITC, fluorescein isothiocyanate; NSF, N-ethylmaleimide sensitive factor; RPMI medium, Rosewell Park Memorial Institute medium; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; SNAP, soluble NSF attachment proteins, SNARE, SNAP receptor. 2
are membrane components which determine the specificity of the docking and fusion of vesicles to the correct target membranes, identified based on their ability to interact with SNAP (20). Genetic dissections of the yeast secretory pathway and the biochemical characterization of molecules involved in synaptic vesicle docking and fusion have resulted in the isolation and/ or molecular cloning of putative SNARE molecules which are structurally related (5–7). SNARES can be broadly classified into vesicle (v-) or target (t-) membrane SNAREs. The prototype tSNAREs are the syntaxin family of proteins. Its first member to be reported, syntaxin 1A, was first characterized as a neuronal specific protein involved in the regulation of neurotransmitter release (6). Its localization to the plasma membrane and its interaction with the synaptic vesicle v-SNARE synaptobrevin point to its function as a t-SNARE. Subsequently, a family of syntaxin-related molecules which are more ubiquitously expressed has been identified (7, 9). Syntaxins 2, 3 and 4 are cell surface proteins (7, 12, 10) whereas syntaxin 5 and syntaxin 6 are localized to the Golgi region (7, 9). Recently, we and others have also identified a syntaxin-like molecule, syntaxin 7, which is localized to the endosome (25, 27). In this report, we present another syntaxin-like molecule which is TGN-localized. MATERIALS AND METHODS Materials. Cell lines were primarily from the American Type Culture Collection. Cell culture media and fetal bovine serum (FBS) were from Gibco BRL. Brefeldin A (BFA) was from Epicentre Technologies. Wortmannin and other common chemicals were mainly from Sigma Chemical Company. FITC or rhodamine-conjugated goat anti-mouse Ig or sheep anti-rabbit Ig were purchased from Boehringer Mannheim Far East (Singapore). The enhanced chemiluminescent (ECL) immunoblot analysis kit and radioisotopes were from Amersham. Expressed sequence tag clones were generated by the Washington University-MERCK EST project, and were obtained from the IMAGE consortium via Research Genetics Inc. (USA).
345
0006-291X/98 $25.00 Copyright q 1998 by Academic Press All rights of reproduction in any form reserved.
AID
BBRC 7966
/
6945$$$441
12-30-97 18:25:53
bbrcgs
AP: BBRC
Vol. 242, No. 2, 1998
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
FIG. 1. Molecular cloning of a novel human syntaxin. (A). The DNA sequence and derived amino acid sequence of the coding region of human syntaxin 10 (hsyn10). The putative transmembrane is boxed. (B). Kyte-Doolittle hydrophilicity plot of the primary sequence of hsyn10. (C). Coils 2.1 analysis of potential coiled-coil domains of hsyn10. The window of analysis is 21 amino acids wide.
Methods. Database searches were performed with the various Basic Local Alignment Search Tools (BLAST) algorithms (1-2) available at the National Center for Biotechnology (NCBI) world wide web server. Library screening, cloning and DNA sequencing were performed using standard methods as described (3). Northern blot analysis was performed using a human multiple tissue Northern (MTN) blot from Clontech (Palo Alto, CA, USA). The cytoplasmic domain of the protein is expressed as a hexahistidine-tagged fusion proteins in bacteria. The fusion proteins were used
to immunize rabbits. Polyclonal antibodies were affinity-purified from serum harvested after several booster injections by the fusion proteins immobilized on nitrocellulose strips. Cells were maintained in RPMI medium supplemented with 10% FBS. Immunofluorescence microscopy was performed as described previously (23-24). Cells plated on coverslips, subjected to various treatments, were fixed with 4% paraformaldehyde followed by sequential incubation with the primary antibodies and FITC or rhodamine-conjugated secondary antibodies. Fluorescence labeling was visualized using
346
AID
BBRC 7966
/
6945$$$442
12-30-97 18:25:53
bbrcgs
AP: BBRC
Vol. 242, No. 2, 1998
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
FIG. 2. (A). The alignment of hsyn10 with rat syntaxin 6 (rsyn6), the yeast Pep12p and human SNAP-25 (hSNAP25) (MegAlign program of DNASTAR). Identical residues in two out of four of the aligned sequences are shaded. The table indicates the percentage similarity and percentage divergence of the sequences compared. (B). The alignment of a region of hsyn10 (amino acids 86 to 148) to rsyn6, EST AA337908the putative human syntaxin 6 and EST W29518-he putative mouse syntaxin 6. In this region, the degree of similarity between hsyn10 and rsyn6 is low but that between rsyn6, W29518 and AA337908 remained very high.
an Axiophot microscope (Carl Zeiss, Inc., Thornwood, NY/USA) with epifluorescence optics or MRC600 (BIORAD) confocal laser optics.
RESULTS AND DISCUSSION Molecular Cloning and Sequencing of a Novel Member of the Syntaxin Family Database searches has allowed us to identified human ESTs (GenBank accession numbers N35629 and W24393)
potentially coding for a novel syntaxin-like molecule. A complete cDNA was isolated from a human pancreas cDNA library and sequencing revealed a 249 amino acid open reading frame as shown in Fig. 1A. The predicted amino acid sequence has a stretch of 22 hydrophobic residues at the C-terminus, as illustrated by a KyteDoolittle hydrophobicity plot (Fig. 1B). This primary structure is characteristic of a hydrophobic tail-anchor.
347
AID
BBRC 7966
/
6945$$$442
12-30-97 18:25:53
bbrcgs
AP: BBRC
Vol. 242, No. 2, 1998
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
FIG. 3. Northern blot analysis of hsyn10. H, heart; B, brain; Pl, placenta; L, lung; Li, liver; SM, skeletal muscle, K, kidney; P, pancreas.
The polypeptide has several potential regions that may form coiled-coil structures, as revealed by the Coils version 2.2 program (Fig. 1C). Based on ESTs identified by database searches, Bock and Scheller have recently presented a series of ten novel syntaxin-like molecules and have numbered them consecutively as syntaxins 7 to 16 (8). The ESTs N35629 and W24393 corresponded to syntaxin 10 and we have therefore retained the name human syntaxin 10 (hsyn10) for our clone.
A database search using the NCBI BLAST program revealed that the coding sequence of has the highest homology with other known SNAREs (Fig. 2A). It is particularly homologous to the recently reported rat syntaxin 6 (rsyn6) (9). Database search results however suggest that the human cDNA is not the human homolog of rat syntaxin 6. BLAST searches with both hsyn10 and rsyn6 revealed a mouse EST (GenBank accession number W29518) with a higher degree of similarity to rsyn6 than hsyn10. This is likely to be the mouse syntaxin 6. Furthermore, a BLAST search with rsyn6 revealed another human EST (GenBank accession number AA337908) which a higher degree of similarity to rsyn6 than hsyn10. This is shown in Fig. 2B for a region of human syntaxin 10 (amino acids 86 to 148) with a particularly low homology to rat syntaxin 6. In this region however, the degree of identity between the putative mouse syntaxin 6 (W29518) and the human EST AA337908 remained very high. It is clear that the EST AA337908 represents the human homolog of rat syntaxin 6. hsyn10 may thus be a closely related isoform of human syntaxin 6. Further support for this notion come from data presented below. A multiple tissue Northern blot with the full length cDNA revealed a single transcript of about 1 kb, which is particularly enriched in the heart, skeletal muscles and pancreas (Fig. 3).
FIG. 4. Indirect immunofluorescence analysis of hsyn10. Cells were fixed with 4% paraformyldehyde and incubated with affinity-purified rabbit polyclonal antibody against hsyn10 (A and E, A431 cells; B, Vero cells; C, normal rat kidney (NRK) cells; D, mouse embryonic fibroblast (MEF) cells). This is followed by incubation with FITC-labeled anti-rabbit IgG. Bar Å 10 mm.
348
AID
BBRC 7966
/
6945$$$442
12-30-97 18:25:53
bbrcgs
AP: BBRC
Vol. 242, No. 2, 1998
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
FIG. 5. The effect of BFA treatment on the distribution of hsyn10. Cells were either untreated (A, B, and E) or treated with 10 mg/ml BFA for 1h (C, D, and F). Cells were then fixed with 4% paraformyldehyde and incubated with either affinity-purified rabbit polyclonal antibody against hsyn10 together with a monoclonal antibody against the Golgi SNARE GS28 (22) (A-D) or with rabbit polyclonal antiserum against b1,4-galactosyltransferase alone (21) (E-F). This is followed by incubation with FITC-labeled anti-rabbit IgG and rhodamine-labeled anti-mouse IgG (A-D) or FITC-labeled anti-rabbit IgG alone (E-F). B, D, E and F: FITC channel; A and C: rhodamine channel. Bar Å 10 mm.
Syntaxin 10 Is Localized to the Trans-Golgi Network To further characterize human syntaxin 10, rabbit polyclonal antibodies were raised using bacterially expressed fusion protein. The affinity-purified antibody detected a Ç36 kDa band by immunoblot analysis of
the in vitro translated product of the full length cDNA and A431 cell lysates (not shown). Detection can be abolished by co- or preincubation of the antibody with excess amount of the fusion protein (not shown). As a first step towards functional characterization of syntaxin 10, we performed indirect immunofluores-
349
AID
BBRC 7966
/
6945$$$442
12-30-97 18:25:53
bbrcgs
AP: BBRC
Vol. 242, No. 2, 1998
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
cence microscopy to localise the protein. As shown in Fig. 4, the affinity-purified antibody labeled intracellular perinuclear structures resembling that of the Golgi apparatus in human (A431 cells, A) and monkey (Vero cells, B) but not rat (NRK cells, C) or mouse (embryonic fibroblast cells, D). No immunoreactivity was observed in several other rat and mouse cell lines, indicating that the hsyn10 antibody is reactive with primate but not rodent cells. To further determine the subcellular localization of hsyn10, we double label for hsyn10 and a Golgi marker, the Golgi SNARE GS28 (22) in cells which are untreated and cells treated with the fungal metabolite brefeldin A (BFA). BFA has varying effects on the morphology of subcellular organelles and on the distribution of various markers on these organelles (11). The distribution of Golgi markers to the endoplasmic reticulum (11, 13) and the collapse of TGN markers TGN38 (15) and furin proprotein convertase (14) into the microtubule organising center (MTOC) has been extensively documented. The effect of BFA on the morphology of a particular protein is therefore often useful in determining its subcellular localization. As shown in Fig. 5, the perinuclear staining of hsyn10 (A) colocalized well with the perinuclear Golgi staining of the Golgi SNARE GS28 (22) (B). Treatment of A431 cells with 10 mg/ml BFA for 1h distributes the Golgi staining of GS28 to the reticular staining of the endoplasmic reticulum (ER) (D). However, this treatment resulted in the collapsed of the perinuclear hsyn10 structure into a compact structure characteristic of the MTOC (C). Similar to GS28, the perinuclear staining of a resident Golgi marker, b1,4-galactosyltransferase (E), is distributed to that of the ER upon BFA treatment (F). Treatment of cells with 1 mM wortmannin for 1h did not alter the morphology of the structures labeled by hsyn10 (not shown). These results, taken together, suggest that the perinuclear staining of syntaxin 10 is not that of the Golgi apparatus or the endosome, but rather that of the TGN. In our case, the lack of a suitable antibody against a TGN marker in primate cells did not allow double-labeling experiments to be performed. In the context of its localization, hsyn10 may function to receive vesicles either from the Golgi stack or from the endosome. The fact that a putative rodent homolog could not be found in the EST database and that hsyn10 antibody does not detect a rodent protein in rodent cells suggest that hsyn10 may be a primate specific isoform. Elucidation of its exact role in transport awaits experiments involving effective functional disruption of the molecule. ACKNOWLEDGMENTS W.H. is supported by a research grant from the Institute of Molecular and Cell Biology. The assistance and participation of Lay Kheng Lim and San San Lee is gratefully acknowledged. We thank Dr. V. N. Subramaniam for antibody against GS28.
REFERENCES 1. Altschul, S. F., Gish, W., Miller, W., Myers, E. W., and Lipman, D. J. (1990) J. Mol. Biol. 215, 403–410. 2. Altschul, S. F., Madden, T. L., Scha¨ffer, A. A., Zhang, J., Zhang, Z., Miller, W., and Lipman, D. J. (1997) Nucleic Acids Res. 25, 3389–3402. 3. Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D., Seidman, J. G., Smith, J. A., and Struhl, K. (Eds.) (1993) Current Protocols in Molecular Biology. Greene Publishing/Wiley Interscience, New York. 4. Barlowe, C., Orci, L., Yeung, T., Hosobuchi, M., Hamamoto, S., Salama, N., Rexach, M. F., Ravazzola, M., Amherdt, M., and Schekman, R. (1994) Cell 77, 895–907. 5. Bennett, M. K., and Scheller, R. H. (1993) Proc. Natl. Acad. Sci. (USA). 90, 2559–2563. 6. Bennett, M. K., Calakos, N., and Scheller, R. H. (1992) Science 257, 255–259. 7. Bennett, M. K., Garcia-Arraras, J. E., Elferink, L. A., Peterson, K., Fleming, A. M., Hazuka, C. D., and Scheller, R. H. (1993) Cell 74, 863–873. 8. Bock, J. B., and Scheller, R. H. (1997) Nature 387, 133–135. 9. Bock, J. B., Lin, R. C., and Scheller, R. H. (1996) J. Biol. Chem. 271, 17961–17965. 10. Gaisano, H. Y., Ghai, M., Malkus, P. N., Sheu, L., Bouquillon, A., Bennett, M. K., and Trimble, W. S. (1996) Mol. Biol. Cell 7, 2019–2027. 11. Klausner, R. D., Donaldson, J. G., and Lippincott-Schwartz, J. (1992) J. Cell Biol. 116, 1071–1080. 12. Low, S. H., Chapin, S. J., Weimbs, T., Ko¨mu¨ves, L. G., Bennett, M. K., and Mostov, K. (1996) Mol. Biol. Cell 7, 2007–2018. 13. Lippincott-Schwartz, J., Yuan, L. C., Tipper, C., Amherdt, M., Orci, L., and Klausner, R. (1991) Cell 67, 601–616. 14. Molloy, S. S., Thomas, L., VanSlyke, J. K., Stenberg, P. E., and Thomas, G. (1994) EMBO J. 13, 18–33. 15. Reaves, B., and Banting, G. (1992) J. Cell Biol. 116, 85–94. 16. Rothman, J. E. (1994) Nature 372, 55–63. 17. Rothman, J. E., and Orci, L. (1992) Nature 355, 409–415. 18. Rothman, J. E., and Warren, G. (1994) Curr. Biol. 4, 220–233. 19. So¨llner, T., Bennet, M. K., Whiteheart, S. W., Scheller, R. H., and Rothman, J. E. (1993) Cell 75, 409–418. 20. So¨llner, T., Whiteheart, S. W., Brunner, M., Erdjument-Bromage, H., Geromanos, S., Tempst, P., and Rothman, J. E. (1993) Nature 362, 318–324. 21. Subramaniam, V. N., Abdul Rashid, b. M. Y., Wong, S. H., Lim, G. B., Chew, M., and Hong, W. (1992) J. Biol. Chem. 267, 12016– 12021. 22. Subramaniam, V. N., Krijnse-Locker, J., Tang, B. L., Ericsson, M., Abdul Rashid, M. Y., Nilsson, T., Griffiths, G., and Hong, W. (1995) J. Cell Sci. 108, 2405–2414. 23. Tang, B. L., Low, S. H., and Hong, W. (1995) Eur. J. Cell Biol. 68, 199–205. 24. Tang, B. L., Peter, F., Krijnse-Locker, J., Low, S. H., Griffiths, G., and Hong, W. (1997) Mol. Cell Biol. 17, 256–266. 25. Wang, H., Frelin, L., and Pevsner, J. (1997) Gene 199, 39–48. 26. Waters, M. G., Serafini, T., and Rothman, J. E. (1991) Nature 349, 248–251 Whitney, J. A., Gomez, M., Sheff, D., Kreis, T. E., and Mellman, I. (1995) Cell 83, 703–713. 27. Wong, S. H., Xu, Y., Zhang, T., and Hong, W. (1998) J. Biol. Chem., in press.
350
AID
BBRC 7966
/
6945$$$442
12-30-97 18:25:53
bbrcgs
AP: BBRC
242, 345–350 (1998)
RC977966
Syntaxin 10: A Member of the Syntaxin Family Localized to the Trans-Golgi Network Bor Luen Tang,1 Delphine Y. H. Low,1 Andrew E. H. Tan, and Wanjin Hong2 Membrane Biology Laboratory, Institute of Molecular and Cell Biology, 30 Medical Drive, Singapore 117609, Republic of Singapore
Received December 1, 1997
We have cloned a new member of the syntaxin family of proteins, designated human syntaxin 10 (hsyn10). The open reading frame encodes a polypeptide of 249 amino acids with potential coiled-coil domains and a carboxy-terminal hydrophobic tail. hsyn10 is particularly homologous to the recently reported rat syntaxin 6 (about 60% identity). Northern blot analysis showed that the transcript is enriched in the heart, skeletal muscles and pancreas. Indirect immunofluorescence studies using polyclonal antibodies raised against recombinant protein showed that the protein is localized to intracellular membrane structures, with perinuclear staining patterns colocalising well with the Golgi SNARE GS28. Morphological alterations of the staining pattern of the protein with brefeldin A but not wortmannin treatment indicate that the protein is localize to the transGolgi network. q 1998 Academic Press
Membranous compartments in eukaryotic cells are linked by vesicular transport processes (16, 17). Molecular components required for both vesicle budding (4, 26) and vesicle docking/fusion processes (16-20) have been isolated. The N-ethylmaleimide sensitive factor (NSF), an ATPase whose activity regulates the formation and dissociation of fusion complexes works in conjunction with another soluble factor, the soluble NSF attachment protein (SNAP), in mediating vesicle docking and/or fusion (17). SNAP receptors, or SNAREs, 1
The first two authors contributed equally. Correspondence. Fax: (65) 779-1117. E-mail: mcbhwj@leonis. nus.sg. Abbreviations used: ER, endoplasmic reticulum; FBS, fetal bovine serum; FITC, fluorescein isothiocyanate; NSF, N-ethylmaleimide sensitive factor; RPMI medium, Rosewell Park Memorial Institute medium; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; SNAP, soluble NSF attachment proteins, SNARE, SNAP receptor. 2
are membrane components which determine the specificity of the docking and fusion of vesicles to the correct target membranes, identified based on their ability to interact with SNAP (20). Genetic dissections of the yeast secretory pathway and the biochemical characterization of molecules involved in synaptic vesicle docking and fusion have resulted in the isolation and/ or molecular cloning of putative SNARE molecules which are structurally related (5–7). SNARES can be broadly classified into vesicle (v-) or target (t-) membrane SNAREs. The prototype tSNAREs are the syntaxin family of proteins. Its first member to be reported, syntaxin 1A, was first characterized as a neuronal specific protein involved in the regulation of neurotransmitter release (6). Its localization to the plasma membrane and its interaction with the synaptic vesicle v-SNARE synaptobrevin point to its function as a t-SNARE. Subsequently, a family of syntaxin-related molecules which are more ubiquitously expressed has been identified (7, 9). Syntaxins 2, 3 and 4 are cell surface proteins (7, 12, 10) whereas syntaxin 5 and syntaxin 6 are localized to the Golgi region (7, 9). Recently, we and others have also identified a syntaxin-like molecule, syntaxin 7, which is localized to the endosome (25, 27). In this report, we present another syntaxin-like molecule which is TGN-localized. MATERIALS AND METHODS Materials. Cell lines were primarily from the American Type Culture Collection. Cell culture media and fetal bovine serum (FBS) were from Gibco BRL. Brefeldin A (BFA) was from Epicentre Technologies. Wortmannin and other common chemicals were mainly from Sigma Chemical Company. FITC or rhodamine-conjugated goat anti-mouse Ig or sheep anti-rabbit Ig were purchased from Boehringer Mannheim Far East (Singapore). The enhanced chemiluminescent (ECL) immunoblot analysis kit and radioisotopes were from Amersham. Expressed sequence tag clones were generated by the Washington University-MERCK EST project, and were obtained from the IMAGE consortium via Research Genetics Inc. (USA).
345
0006-291X/98 $25.00 Copyright q 1998 by Academic Press All rights of reproduction in any form reserved.
AID
BBRC 7966
/
6945$$$441
12-30-97 18:25:53
bbrcgs
AP: BBRC
Vol. 242, No. 2, 1998
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
FIG. 1. Molecular cloning of a novel human syntaxin. (A). The DNA sequence and derived amino acid sequence of the coding region of human syntaxin 10 (hsyn10). The putative transmembrane is boxed. (B). Kyte-Doolittle hydrophilicity plot of the primary sequence of hsyn10. (C). Coils 2.1 analysis of potential coiled-coil domains of hsyn10. The window of analysis is 21 amino acids wide.
Methods. Database searches were performed with the various Basic Local Alignment Search Tools (BLAST) algorithms (1-2) available at the National Center for Biotechnology (NCBI) world wide web server. Library screening, cloning and DNA sequencing were performed using standard methods as described (3). Northern blot analysis was performed using a human multiple tissue Northern (MTN) blot from Clontech (Palo Alto, CA, USA). The cytoplasmic domain of the protein is expressed as a hexahistidine-tagged fusion proteins in bacteria. The fusion proteins were used
to immunize rabbits. Polyclonal antibodies were affinity-purified from serum harvested after several booster injections by the fusion proteins immobilized on nitrocellulose strips. Cells were maintained in RPMI medium supplemented with 10% FBS. Immunofluorescence microscopy was performed as described previously (23-24). Cells plated on coverslips, subjected to various treatments, were fixed with 4% paraformaldehyde followed by sequential incubation with the primary antibodies and FITC or rhodamine-conjugated secondary antibodies. Fluorescence labeling was visualized using
346
AID
BBRC 7966
/
6945$$$442
12-30-97 18:25:53
bbrcgs
AP: BBRC
Vol. 242, No. 2, 1998
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
FIG. 2. (A). The alignment of hsyn10 with rat syntaxin 6 (rsyn6), the yeast Pep12p and human SNAP-25 (hSNAP25) (MegAlign program of DNASTAR). Identical residues in two out of four of the aligned sequences are shaded. The table indicates the percentage similarity and percentage divergence of the sequences compared. (B). The alignment of a region of hsyn10 (amino acids 86 to 148) to rsyn6, EST AA337908the putative human syntaxin 6 and EST W29518-he putative mouse syntaxin 6. In this region, the degree of similarity between hsyn10 and rsyn6 is low but that between rsyn6, W29518 and AA337908 remained very high.
an Axiophot microscope (Carl Zeiss, Inc., Thornwood, NY/USA) with epifluorescence optics or MRC600 (BIORAD) confocal laser optics.
RESULTS AND DISCUSSION Molecular Cloning and Sequencing of a Novel Member of the Syntaxin Family Database searches has allowed us to identified human ESTs (GenBank accession numbers N35629 and W24393)
potentially coding for a novel syntaxin-like molecule. A complete cDNA was isolated from a human pancreas cDNA library and sequencing revealed a 249 amino acid open reading frame as shown in Fig. 1A. The predicted amino acid sequence has a stretch of 22 hydrophobic residues at the C-terminus, as illustrated by a KyteDoolittle hydrophobicity plot (Fig. 1B). This primary structure is characteristic of a hydrophobic tail-anchor.
347
AID
BBRC 7966
/
6945$$$442
12-30-97 18:25:53
bbrcgs
AP: BBRC
Vol. 242, No. 2, 1998
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
FIG. 3. Northern blot analysis of hsyn10. H, heart; B, brain; Pl, placenta; L, lung; Li, liver; SM, skeletal muscle, K, kidney; P, pancreas.
The polypeptide has several potential regions that may form coiled-coil structures, as revealed by the Coils version 2.2 program (Fig. 1C). Based on ESTs identified by database searches, Bock and Scheller have recently presented a series of ten novel syntaxin-like molecules and have numbered them consecutively as syntaxins 7 to 16 (8). The ESTs N35629 and W24393 corresponded to syntaxin 10 and we have therefore retained the name human syntaxin 10 (hsyn10) for our clone.
A database search using the NCBI BLAST program revealed that the coding sequence of has the highest homology with other known SNAREs (Fig. 2A). It is particularly homologous to the recently reported rat syntaxin 6 (rsyn6) (9). Database search results however suggest that the human cDNA is not the human homolog of rat syntaxin 6. BLAST searches with both hsyn10 and rsyn6 revealed a mouse EST (GenBank accession number W29518) with a higher degree of similarity to rsyn6 than hsyn10. This is likely to be the mouse syntaxin 6. Furthermore, a BLAST search with rsyn6 revealed another human EST (GenBank accession number AA337908) which a higher degree of similarity to rsyn6 than hsyn10. This is shown in Fig. 2B for a region of human syntaxin 10 (amino acids 86 to 148) with a particularly low homology to rat syntaxin 6. In this region however, the degree of identity between the putative mouse syntaxin 6 (W29518) and the human EST AA337908 remained very high. It is clear that the EST AA337908 represents the human homolog of rat syntaxin 6. hsyn10 may thus be a closely related isoform of human syntaxin 6. Further support for this notion come from data presented below. A multiple tissue Northern blot with the full length cDNA revealed a single transcript of about 1 kb, which is particularly enriched in the heart, skeletal muscles and pancreas (Fig. 3).
FIG. 4. Indirect immunofluorescence analysis of hsyn10. Cells were fixed with 4% paraformyldehyde and incubated with affinity-purified rabbit polyclonal antibody against hsyn10 (A and E, A431 cells; B, Vero cells; C, normal rat kidney (NRK) cells; D, mouse embryonic fibroblast (MEF) cells). This is followed by incubation with FITC-labeled anti-rabbit IgG. Bar Å 10 mm.
348
AID
BBRC 7966
/
6945$$$442
12-30-97 18:25:53
bbrcgs
AP: BBRC
Vol. 242, No. 2, 1998
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
FIG. 5. The effect of BFA treatment on the distribution of hsyn10. Cells were either untreated (A, B, and E) or treated with 10 mg/ml BFA for 1h (C, D, and F). Cells were then fixed with 4% paraformyldehyde and incubated with either affinity-purified rabbit polyclonal antibody against hsyn10 together with a monoclonal antibody against the Golgi SNARE GS28 (22) (A-D) or with rabbit polyclonal antiserum against b1,4-galactosyltransferase alone (21) (E-F). This is followed by incubation with FITC-labeled anti-rabbit IgG and rhodamine-labeled anti-mouse IgG (A-D) or FITC-labeled anti-rabbit IgG alone (E-F). B, D, E and F: FITC channel; A and C: rhodamine channel. Bar Å 10 mm.
Syntaxin 10 Is Localized to the Trans-Golgi Network To further characterize human syntaxin 10, rabbit polyclonal antibodies were raised using bacterially expressed fusion protein. The affinity-purified antibody detected a Ç36 kDa band by immunoblot analysis of
the in vitro translated product of the full length cDNA and A431 cell lysates (not shown). Detection can be abolished by co- or preincubation of the antibody with excess amount of the fusion protein (not shown). As a first step towards functional characterization of syntaxin 10, we performed indirect immunofluores-
349
AID
BBRC 7966
/
6945$$$442
12-30-97 18:25:53
bbrcgs
AP: BBRC
Vol. 242, No. 2, 1998
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
cence microscopy to localise the protein. As shown in Fig. 4, the affinity-purified antibody labeled intracellular perinuclear structures resembling that of the Golgi apparatus in human (A431 cells, A) and monkey (Vero cells, B) but not rat (NRK cells, C) or mouse (embryonic fibroblast cells, D). No immunoreactivity was observed in several other rat and mouse cell lines, indicating that the hsyn10 antibody is reactive with primate but not rodent cells. To further determine the subcellular localization of hsyn10, we double label for hsyn10 and a Golgi marker, the Golgi SNARE GS28 (22) in cells which are untreated and cells treated with the fungal metabolite brefeldin A (BFA). BFA has varying effects on the morphology of subcellular organelles and on the distribution of various markers on these organelles (11). The distribution of Golgi markers to the endoplasmic reticulum (11, 13) and the collapse of TGN markers TGN38 (15) and furin proprotein convertase (14) into the microtubule organising center (MTOC) has been extensively documented. The effect of BFA on the morphology of a particular protein is therefore often useful in determining its subcellular localization. As shown in Fig. 5, the perinuclear staining of hsyn10 (A) colocalized well with the perinuclear Golgi staining of the Golgi SNARE GS28 (22) (B). Treatment of A431 cells with 10 mg/ml BFA for 1h distributes the Golgi staining of GS28 to the reticular staining of the endoplasmic reticulum (ER) (D). However, this treatment resulted in the collapsed of the perinuclear hsyn10 structure into a compact structure characteristic of the MTOC (C). Similar to GS28, the perinuclear staining of a resident Golgi marker, b1,4-galactosyltransferase (E), is distributed to that of the ER upon BFA treatment (F). Treatment of cells with 1 mM wortmannin for 1h did not alter the morphology of the structures labeled by hsyn10 (not shown). These results, taken together, suggest that the perinuclear staining of syntaxin 10 is not that of the Golgi apparatus or the endosome, but rather that of the TGN. In our case, the lack of a suitable antibody against a TGN marker in primate cells did not allow double-labeling experiments to be performed. In the context of its localization, hsyn10 may function to receive vesicles either from the Golgi stack or from the endosome. The fact that a putative rodent homolog could not be found in the EST database and that hsyn10 antibody does not detect a rodent protein in rodent cells suggest that hsyn10 may be a primate specific isoform. Elucidation of its exact role in transport awaits experiments involving effective functional disruption of the molecule. ACKNOWLEDGMENTS W.H. is supported by a research grant from the Institute of Molecular and Cell Biology. The assistance and participation of Lay Kheng Lim and San San Lee is gratefully acknowledged. We thank Dr. V. N. Subramaniam for antibody against GS28.
REFERENCES 1. Altschul, S. F., Gish, W., Miller, W., Myers, E. W., and Lipman, D. J. (1990) J. Mol. Biol. 215, 403–410. 2. Altschul, S. F., Madden, T. L., Scha¨ffer, A. A., Zhang, J., Zhang, Z., Miller, W., and Lipman, D. J. (1997) Nucleic Acids Res. 25, 3389–3402. 3. Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D., Seidman, J. G., Smith, J. A., and Struhl, K. (Eds.) (1993) Current Protocols in Molecular Biology. Greene Publishing/Wiley Interscience, New York. 4. Barlowe, C., Orci, L., Yeung, T., Hosobuchi, M., Hamamoto, S., Salama, N., Rexach, M. F., Ravazzola, M., Amherdt, M., and Schekman, R. (1994) Cell 77, 895–907. 5. Bennett, M. K., and Scheller, R. H. (1993) Proc. Natl. Acad. Sci. (USA). 90, 2559–2563. 6. Bennett, M. K., Calakos, N., and Scheller, R. H. (1992) Science 257, 255–259. 7. Bennett, M. K., Garcia-Arraras, J. E., Elferink, L. A., Peterson, K., Fleming, A. M., Hazuka, C. D., and Scheller, R. H. (1993) Cell 74, 863–873. 8. Bock, J. B., and Scheller, R. H. (1997) Nature 387, 133–135. 9. Bock, J. B., Lin, R. C., and Scheller, R. H. (1996) J. Biol. Chem. 271, 17961–17965. 10. Gaisano, H. Y., Ghai, M., Malkus, P. N., Sheu, L., Bouquillon, A., Bennett, M. K., and Trimble, W. S. (1996) Mol. Biol. Cell 7, 2019–2027. 11. Klausner, R. D., Donaldson, J. G., and Lippincott-Schwartz, J. (1992) J. Cell Biol. 116, 1071–1080. 12. Low, S. H., Chapin, S. J., Weimbs, T., Ko¨mu¨ves, L. G., Bennett, M. K., and Mostov, K. (1996) Mol. Biol. Cell 7, 2007–2018. 13. Lippincott-Schwartz, J., Yuan, L. C., Tipper, C., Amherdt, M., Orci, L., and Klausner, R. (1991) Cell 67, 601–616. 14. Molloy, S. S., Thomas, L., VanSlyke, J. K., Stenberg, P. E., and Thomas, G. (1994) EMBO J. 13, 18–33. 15. Reaves, B., and Banting, G. (1992) J. Cell Biol. 116, 85–94. 16. Rothman, J. E. (1994) Nature 372, 55–63. 17. Rothman, J. E., and Orci, L. (1992) Nature 355, 409–415. 18. Rothman, J. E., and Warren, G. (1994) Curr. Biol. 4, 220–233. 19. So¨llner, T., Bennet, M. K., Whiteheart, S. W., Scheller, R. H., and Rothman, J. E. (1993) Cell 75, 409–418. 20. So¨llner, T., Whiteheart, S. W., Brunner, M., Erdjument-Bromage, H., Geromanos, S., Tempst, P., and Rothman, J. E. (1993) Nature 362, 318–324. 21. Subramaniam, V. N., Abdul Rashid, b. M. Y., Wong, S. H., Lim, G. B., Chew, M., and Hong, W. (1992) J. Biol. Chem. 267, 12016– 12021. 22. Subramaniam, V. N., Krijnse-Locker, J., Tang, B. L., Ericsson, M., Abdul Rashid, M. Y., Nilsson, T., Griffiths, G., and Hong, W. (1995) J. Cell Sci. 108, 2405–2414. 23. Tang, B. L., Low, S. H., and Hong, W. (1995) Eur. J. Cell Biol. 68, 199–205. 24. Tang, B. L., Peter, F., Krijnse-Locker, J., Low, S. H., Griffiths, G., and Hong, W. (1997) Mol. Cell Biol. 17, 256–266. 25. Wang, H., Frelin, L., and Pevsner, J. (1997) Gene 199, 39–48. 26. Waters, M. G., Serafini, T., and Rothman, J. E. (1991) Nature 349, 248–251 Whitney, J. A., Gomez, M., Sheff, D., Kreis, T. E., and Mellman, I. (1995) Cell 83, 703–713. 27. Wong, S. H., Xu, Y., Zhang, T., and Hong, W. (1998) J. Biol. Chem., in press.
350
AID
BBRC 7966
/
6945$$$442
12-30-97 18:25:53
bbrcgs
AP: BBRC