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Syntaxin 11: A Member of the Syntaxin Family without a Carboxyl Terminal Transmembrane Domain
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS ARTICLE NO.
245, 627–632 (1998)
RC988490
Syntaxin 11: A Member of the Syntaxin Family without a Carboxyl Terminal Transmembrane Domain Bor Luen Tang, Delphine Y. H. Low, and Wanjin Hong1 Membrane Biology Laboratory, Institute of Molecular and Cell Biology, 30 Medical Drive, Singapore 117609, Republic of Singapore
Received March 3, 1998
We have cloned a novel syntaxin-like molecule, designated human syntaxin 11 (hsyn11). The open reading frame encodes a polypeptide of 287 amino acids with potential coiled-coil domains. hsyn11 has extensive homology to members of the syntaxin family, particularly syntaxin 1 and syntaxin 2. Unlike other members of the syntaxin family, however, hsyn11 has a short cysteinerich carboxyl-terminal tail but not a typical hydrophobic domain which may serve as a membrane anchor. Northern blot analysis revealed two transcripts of Ç0.8 kb and Ç1.7 kb in length that are particularly abundant in heart and placenta, although lower levels were also detectable in other tissues except in the brain. Consistent with the lack of a distinct membrane anchorage sequence in hsyn11, indirect immunofluorescence microscopy of transiently expressed N-terminally myc-tagged hsyn11 revealed a diffuse, cytoplasmic labeling. q 1998 Academic Press Key Words: syntaxin; tail anchor; protein transport.
Trafficking between intracellular membranous compartments is largely mediated by vesicular transport processes. High degrees of specificity and complexity are exerted in the regulations of both vesicle budding and vesicle docking/fusion. The docking and fusion processes of transport vesicles requires the concerted action of the cytosolic N-ethylmaleimide sensitive factor (NSF), an ATPase whose activity regulates the formation and dissociation of fusion complexes and another soluble factor, the soluble NSF attachment protein (SNAP) (17-18). These cytosolic factors, though essential, are insufficient to determine the specificity of the docking and fusion of vesicles to the correct target membranes. Membrane 1 Corresponding author. Fax: (65) 779-1117. E-mail: mcbhwj@ imcb.nus.edu.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.
components which determine the specificity of vectorial transport were subsequently identified based on their interaction with SNAP (20-21), and are therefore known as SNAP receptors (SNAREs). Initially thought to be localized exclusively in their respective membrane compartments in accordance with their function, SNAREs were broadly classified into vesicle (v-) or target (t-) membrane SNAREs. Recent reports however suggest that both v- and tSNAREs can exist as a complex on synaptic vesicles (15) as well as endoplasmic reticulum-derived transport vesicles (19). Thus NSF, originally thought to be triggering vesicle fusion, appear to act at an earlier step in priming of SNAREs for docking (15, 28). 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 SNAREs which are structurally related (4-7). Syntaxin 1A was first characterized as a neuronal specific protein involved in the regulation of neurotransmitter release (5). Its localization to the plasma membrane and its interaction with the synaptic vesicle v-SNARE synaptobrevin/VAMP-1 point to its function as a tSNARE. Subsequently, a family of syntaxin-related proteins which are more ubiquitously expressed has been identified (6, 8). Syntaxins 2, 3 and 4 are cell surface proteins (6, 9, 12), syntaxin 5 and 6 are localized to the Golgi region (6, 8) and syntaxin 7 is localized to the endosome (29-30). We have also recently reported the cloning of syntaxin 10 which is localized to the trans-Golgi network (23), syntaxin 12 which appears to be endosomal (27) and syntaxin 16, whose myctagged form is localized to the Golgi apparatus (24). A characteristic feature of the syntaxin family of proteins is presence of a carboxyl terminal hydrophobic domain which anchors the protein to the membrane. This is also a feature shared by other SNAREs (4). There are, however, members of the SNARE family which does not have a carboxyl terminal anchor, such as the neuronal SNAP-25 and its more ubiquitous homologue, SNAP-23 (16). These molecules are palmitoyl-
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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 11 (hsyn11). B). Kyte-Doolittle hydrophilicity plot of the primary sequence of hsyn11. C). Coils 2.1 analysis of potential coiled-coil domains of hsyn11. The window of analysis is 21 amino acids wide.
ated, which allows them to be membrane associated (10, 16). The yeast protein Ykt6p and its human homolog, which functions in endoplasmic reticulum (ER)Golgi transport, also lacks a membrane anchor and are prenylated (14). We present in this report the first member of the syntaxin family which lacks a distinct membrane spanning domain at the carboxyl terminus.
MATERIALS AND METHODS Materials. Cell lines were primarily obtained from the American Type Culture Collection. Cell culture media and fetal bovine serum (FBS) were from Gibco BRL. FITC or rhodamine-conjugated goat antimouse Ig or sheep anti-rabbit Ig were purchased from Boehringer Mannheim Far East (Singapore). Expressed sequence tag clones were generated by the Washington University-MERCK EST project, and were ob-
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FIG. 2. The alignment of hsyn11 with human syntaxin 1A (hsyn1A), human syntaxin 1B (hsyn1B), human syntaxin 2 (hsyn2), human syntaxin 3 (hsyn3) and human syntaxin 4 (hsyn4) (MegAlign program of DNASTAR). Residues identical to hsyn11 are shaded. The table indicates the percentage similarity and percentage divergence of the sequences compared. tained from the IMAGE consortium via Research Genetics Inc. (USA). Human heart cDNA library was from Strategene (USA). Ribophorin antiserum is kindly provided by Dr David Meyer (University of California, Los Angeles, CA/USA) (11). Rabbit polyclonal antibodies against syntaxin 5 (22) were kindly provided by Dr. Nathan Subramaniam. Methods. Database searches were performed with the various Basic Local Alignment Search Tools (BLAST) algorithms (1-2) avail-
able at the National Center for Biotechnology (NCBI) world wide web server. Coils 2.1 analysis (13) was performed using the Coils 2.1 program available at the ISREC Bioinformatics Group 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. Transient transfection was performed with Lipofectamine (Gibco
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FIG. 3. Northern blot analysis of hsyn11. H, heart; B, brain; Pl, placenta; L, lung; Li, liver; SM, skeletal muscle; K, kidney; P, pancreas.
BRL) according to the manufacturer’s protocol. Cells were maintained in RPMI medium supplemented with 10% FBS. Immunofluorescence microscopy was performed as described previously (25-26). 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 an Axiophot microscope (Carl Zeiss, Inc., Thornwood, NY/USA) with epifluorescence optics or MRC600 (BIORAD) confocal laser optics.
RESULTS A Novel Member of the Syntaxin Family Database searches have allowed us to identified human ESTs (GenBank accession numbers AA227632 and AA262151) potentially coding for a novel syntaxinlike molecule. DNA sequencing of both clones revealed a 287 amino acid open reading frame with extensive homology to other members of the syntaxin family. The predicted protein sequence has a cysteine-rich se-
quence at the C-terminal end as shown in Fig. 1A. The predicted amino acid sequence, however, does not contain a stretch of hydrophobic residues long enough to serve as a putative transmembrane domain, as illustrated by a Kyte-Doolittle hydrophobicity plot (Fig. 1B). Both the EST clones are from the same library. Since it is unusual for a syntaxin-like molecule to be without a sufficiently long stretch of hydrophobic residues at the carboxyl terminus to serve as a membrane anchor, we sought to further confirm the cDNA sequence by sequencing clones from an independent source, a human heart cDNA library (Strategene). Sequencing of three independent clones from this library revealed that they are identical to the sequences of the ESTs. The polypeptide has several potential regions that may form coiled-coil structures, as revealed by the Coils version 2.1 program (Fig. 1C). Bock and Scheller (7) have recently presented a series of ten novel syntaxinlike molecules and have numbered them consecutively as syntaxins 7 to 16 (7). The ESTs AA227632 and AA262151 corresponded to syntaxin 11 and we have thus retained the name human syntaxin 11 (hsyn11). A database search using the NCBI BLAST program revealed that the derived amino acid sequence of hsyn 11 has the highest homology with syntaxin 1, syntaxin 2, syntaxin 3 and syntaxin 4. The alignment of these sequences with hsyn11 is shown in Fig.2. Probing of a multiple tissue Northern blot revealed two transcripts of about 0.8 kb and 1.7 kb, which are particularly enriched in the heart and placenta (Fig.3). Interestingly, the transcript is of low abundance in brain such that it remained undetectable even after longer exposure of the blot. Myc-Tagged hsyn 11 Appeared to Be Cytoplasmic and Not Associated with Endomembranes All members of the syntaxin family identified to date have a stretch of hydrophobic amino acid residues at the C-terminus which serves as a membrane anchor. For hsyn11, this corresponding hydrophobic stretch does not exist. However, hsyn11 could still be tethered to a particular endomembrane through other mechanisms. To investigate this possibility, a myc epitope tag is attached to the N-terminus of hsyn11 (hsyn11-myc) and the subcellular localization of hsyn11-myc is determined by indirect immunofluorescence. As shown in Fig.4, transiently expressed hsyn11-myc in Vero (A, C and E) and COS cells (G) assumed a diffuse, cytoplasmic appearance. Spots of intense labeling that
FIG. 4. Analysis of transiently expressed myc-tagged hsyn11 (hsyn11-myc) by indirect immunofluorescence microscopy. Cells were fixed with 4% paraformyldehyde and incubated with anti-myc monoclonal antibody and rabbit polyclonal antibody against syntaxin 5 (A-B, GH) or rabbit antisera against ribophorin 1 (C, D, E and F). This is followed by incubation with FITC-labeled anti-mouse IgG and rhodaminelabeled anti-rabbit IgG. E and F is a 51 magnified view of the corresponding regions marked by * in C and D (to illustrate the difference in the morphology of the labeling). A, C, E and G - FITC channel; B, D, F and H - rhodamine channel. Note that in the case of the transfected cells in the rhodamine channels, there is a bleed through from some of the bright staining spots labeled with FITC. Bar Å 10 mm. 630
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stand out against a homogenous background probably represents cytoplasmic aggregates of the protein, not unexpected in view of the propensity of hsyn11 to form coiled-coil structures. There were no apparent structures which would suggest association of hsyn11-myc with the Golgi apparatus marked by syntaxin 5 (B) or the ER marked by ribophorin 1 (D). A magnified portion of the cytoplasm of a transfected cell clearly showed the difference between the reticular and punctate staining of the ER ribophorin 1 (F) and the homogenous labeling of hsyn11-myc (E). Finally, transient overexpression of hsyn11-myc in COS cells (G) appeared to disrupt the perinuclear labeling of the Golgi apparatus by syntaxin 5 (H). DISCUSSION The length of the sequence of hydrophobic residues which constitute the tail anchor of members of the syntaxin family typically exceeds 20 amino acids. The shortest amongst them is 17 amino acid residues in length, in the case of syntaxin 5 (6). In marked contrast, hsyn11 does not have a C-terminal hydrophobic domain of significant length. Consistent with this observation, transiently expressed hsyn11-myc appeared to be cytoplasmic and not associated with endomembranes. Beyond the syntaxins, there exist several members of the SNARE superfamily without a hydrophobic tail anchor. The neuronal SNAP25 and its more ubiquitous homolog SNAP23 are associated with membranes as a result of palmitoylation (10, 15). SNAP25 can be found in a preformed ternary complex with synaptobrevin and syntaxin in synaptic vesicles (14). Therefore, SNARE-SNARE association via coiled-coil structures may also serve to tether anchorless SNARE molecules to the membrane. Thus far, several epitope-tagged syntaxins have been faithfully localized to their respective compartments, as confirmed by subsequent availability of their respective antibodies (6, 12, 23, 27). Assuming that the myc-tag did not interfere with membrane association, the apparent cytoplasmic staining of hsyn11myc may be a result of overexpression, particularly if only a small number of membrane association sites exists. Otherwise, non-association of hsyn11-myc with endomembranes would suggests that, if hsyn11 has a role in vesicle trafficking, it functions in an nonconventional manner. ACKNOWLEDGMENT W.H. is supported by a research grant from the Institute of Molecular and Cell Biology.
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 Publ. & Wiley Interscience, New York. 4. Bennett, M. K., and Scheller, R. H. (1993) Proc. Natl. Acad. Sci. (USA) 90, 2559–2563. 5. Bennett, M. K., Calakos, N., and Scheller, R. H. (1992) Science 257, 255–259. 6. 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. 7. Bock, J. B., and Scheller, R. H. (1997) Nature 387, 133–135. 8. Bock, J. B., Lin, R. C., and Scheller, R. H. (1996) J. Biol. Chem. 271, 17961–17965. 9. 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. 10. Hess, D. T., Slater, T. M., Wilson, M. C., and Skene, J. H. P. (1992) J. Neurosci. 12, 4634–4641. 11. Hortsch, M. D., and Meyer, D. I. (1985) Eur. J. Biochem. 150, 559–564. 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. Lupas, A., Van Dyke, M., and Stock, J. (1991) Science 252, 1162– 1164. 14. McNew, J. A., Sogaard, M., Lampen, N. M., Machida, S., Ye, R. R., Lacomis, L., Tempst, P., Rothman, J. E., and Sollner, T. H. (1997) J. Biol. Chem. 272, 17776–17783. 15. Otto, H., Hanson, P. I., and Jahn, R. (1997) Proc. Natl. Acad. Sci. USA 94, 6197–6201. 16. Ravichandran, V., Chawla, A., and Roche, P. A. (1996) J. Biol. Chem. 271, 13300–13303. 17. Rothman, J. E. (1994) Nature 372, 55–63. 18. Rothman, J. E., and Warren, G. (1994) Curr. Biol. 4, 220–233. 19. Rowe, T., Dascher, C., Bannykh, S., Plutner, H., and Balch, W. E. (1998) Science 279, 696–700. 20. So¨llner, T., Bennet, M. K., Whiteheart, S. W., Scheller, R. H., and Rothman, J. E. (1993) Cell 75, 409–418. 21. So¨llner, T., Whiteheart, S. W., Brunner, M., Erdjument-Bromage, H., Geromanos, S., Tempst, P., and Rothman, J. E. (1993) Nature 362, 318–324. 22. Subramaniam, V. N., Loh, E., and Hong, W. (1997) J. Biol. Chem. 272, 25441–25444. 23. Tang, B. L., Low, D. Y. H., Lee, S. S., Tan, A. E. H., and Hong, W. (1998) Biochem. Biophys. Res. Commun. 242, 673–679. 24. Tang, B. L., Low, D. Y. H., Tan, A. E. H., and Hong, W. (1998) Biochem. Biophys. Res. Commun. 242, 345–350. 25. Tang, B. L., Low, S. H., and Hong, W. (1995) Eur. J. Cell Biol. 68, 199–205. 26. Tang, B. L., Peter, F., Krijnse-Locker, J., Low, S. H., Griffiths, G., and Hong, W. (1997) Mol. Cell Biol. 17, 256–266. 27. Tang, B. L., Tan, A. E. H., Lim, L. K., Lee, S. S., Low, D. Y. H., and Hong, W. (1998) J. Biol. Chem. (in press). 28. Ungermann, C., Nichols, B. J., Pelham, H. R., and Wickner, W. (1998) J. Cell Biol. 140, 61–69. 29. Wang, H., Frelin, L., and Pevsner, J. (1997) Gene 199, 39–48. 30. Wong, S. H., Xu, Y., Zhang, T., and Hong, W. (1997) J. Biol. Chem. 273, 375–403.
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Syntaxin 11: A Member of the Syntaxin Family without a Carboxyl Terminal Transmembrane Domain Bor Luen Tang, Delphine Y. H. Low, and Wanjin Hong1 Membrane Biology Laboratory, Institute of Molecular and Cell Biology, 30 Medical Drive, Singapore 117609, Republic of Singapore
Received March 3, 1998
We have cloned a novel syntaxin-like molecule, designated human syntaxin 11 (hsyn11). The open reading frame encodes a polypeptide of 287 amino acids with potential coiled-coil domains. hsyn11 has extensive homology to members of the syntaxin family, particularly syntaxin 1 and syntaxin 2. Unlike other members of the syntaxin family, however, hsyn11 has a short cysteinerich carboxyl-terminal tail but not a typical hydrophobic domain which may serve as a membrane anchor. Northern blot analysis revealed two transcripts of Ç0.8 kb and Ç1.7 kb in length that are particularly abundant in heart and placenta, although lower levels were also detectable in other tissues except in the brain. Consistent with the lack of a distinct membrane anchorage sequence in hsyn11, indirect immunofluorescence microscopy of transiently expressed N-terminally myc-tagged hsyn11 revealed a diffuse, cytoplasmic labeling. q 1998 Academic Press Key Words: syntaxin; tail anchor; protein transport.
Trafficking between intracellular membranous compartments is largely mediated by vesicular transport processes. High degrees of specificity and complexity are exerted in the regulations of both vesicle budding and vesicle docking/fusion. The docking and fusion processes of transport vesicles requires the concerted action of the cytosolic N-ethylmaleimide sensitive factor (NSF), an ATPase whose activity regulates the formation and dissociation of fusion complexes and another soluble factor, the soluble NSF attachment protein (SNAP) (17-18). These cytosolic factors, though essential, are insufficient to determine the specificity of the docking and fusion of vesicles to the correct target membranes. Membrane 1 Corresponding author. Fax: (65) 779-1117. E-mail: mcbhwj@ imcb.nus.edu.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.
components which determine the specificity of vectorial transport were subsequently identified based on their interaction with SNAP (20-21), and are therefore known as SNAP receptors (SNAREs). Initially thought to be localized exclusively in their respective membrane compartments in accordance with their function, SNAREs were broadly classified into vesicle (v-) or target (t-) membrane SNAREs. Recent reports however suggest that both v- and tSNAREs can exist as a complex on synaptic vesicles (15) as well as endoplasmic reticulum-derived transport vesicles (19). Thus NSF, originally thought to be triggering vesicle fusion, appear to act at an earlier step in priming of SNAREs for docking (15, 28). 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 SNAREs which are structurally related (4-7). Syntaxin 1A was first characterized as a neuronal specific protein involved in the regulation of neurotransmitter release (5). Its localization to the plasma membrane and its interaction with the synaptic vesicle v-SNARE synaptobrevin/VAMP-1 point to its function as a tSNARE. Subsequently, a family of syntaxin-related proteins which are more ubiquitously expressed has been identified (6, 8). Syntaxins 2, 3 and 4 are cell surface proteins (6, 9, 12), syntaxin 5 and 6 are localized to the Golgi region (6, 8) and syntaxin 7 is localized to the endosome (29-30). We have also recently reported the cloning of syntaxin 10 which is localized to the trans-Golgi network (23), syntaxin 12 which appears to be endosomal (27) and syntaxin 16, whose myctagged form is localized to the Golgi apparatus (24). A characteristic feature of the syntaxin family of proteins is presence of a carboxyl terminal hydrophobic domain which anchors the protein to the membrane. This is also a feature shared by other SNAREs (4). There are, however, members of the SNARE family which does not have a carboxyl terminal anchor, such as the neuronal SNAP-25 and its more ubiquitous homologue, SNAP-23 (16). These molecules are palmitoyl-
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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 11 (hsyn11). B). Kyte-Doolittle hydrophilicity plot of the primary sequence of hsyn11. C). Coils 2.1 analysis of potential coiled-coil domains of hsyn11. The window of analysis is 21 amino acids wide.
ated, which allows them to be membrane associated (10, 16). The yeast protein Ykt6p and its human homolog, which functions in endoplasmic reticulum (ER)Golgi transport, also lacks a membrane anchor and are prenylated (14). We present in this report the first member of the syntaxin family which lacks a distinct membrane spanning domain at the carboxyl terminus.
MATERIALS AND METHODS Materials. Cell lines were primarily obtained from the American Type Culture Collection. Cell culture media and fetal bovine serum (FBS) were from Gibco BRL. FITC or rhodamine-conjugated goat antimouse Ig or sheep anti-rabbit Ig were purchased from Boehringer Mannheim Far East (Singapore). Expressed sequence tag clones were generated by the Washington University-MERCK EST project, and were ob-
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FIG. 2. The alignment of hsyn11 with human syntaxin 1A (hsyn1A), human syntaxin 1B (hsyn1B), human syntaxin 2 (hsyn2), human syntaxin 3 (hsyn3) and human syntaxin 4 (hsyn4) (MegAlign program of DNASTAR). Residues identical to hsyn11 are shaded. The table indicates the percentage similarity and percentage divergence of the sequences compared. tained from the IMAGE consortium via Research Genetics Inc. (USA). Human heart cDNA library was from Strategene (USA). Ribophorin antiserum is kindly provided by Dr David Meyer (University of California, Los Angeles, CA/USA) (11). Rabbit polyclonal antibodies against syntaxin 5 (22) were kindly provided by Dr. Nathan Subramaniam. Methods. Database searches were performed with the various Basic Local Alignment Search Tools (BLAST) algorithms (1-2) avail-
able at the National Center for Biotechnology (NCBI) world wide web server. Coils 2.1 analysis (13) was performed using the Coils 2.1 program available at the ISREC Bioinformatics Group 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. Transient transfection was performed with Lipofectamine (Gibco
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FIG. 3. Northern blot analysis of hsyn11. H, heart; B, brain; Pl, placenta; L, lung; Li, liver; SM, skeletal muscle; K, kidney; P, pancreas.
BRL) according to the manufacturer’s protocol. Cells were maintained in RPMI medium supplemented with 10% FBS. Immunofluorescence microscopy was performed as described previously (25-26). 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 an Axiophot microscope (Carl Zeiss, Inc., Thornwood, NY/USA) with epifluorescence optics or MRC600 (BIORAD) confocal laser optics.
RESULTS A Novel Member of the Syntaxin Family Database searches have allowed us to identified human ESTs (GenBank accession numbers AA227632 and AA262151) potentially coding for a novel syntaxinlike molecule. DNA sequencing of both clones revealed a 287 amino acid open reading frame with extensive homology to other members of the syntaxin family. The predicted protein sequence has a cysteine-rich se-
quence at the C-terminal end as shown in Fig. 1A. The predicted amino acid sequence, however, does not contain a stretch of hydrophobic residues long enough to serve as a putative transmembrane domain, as illustrated by a Kyte-Doolittle hydrophobicity plot (Fig. 1B). Both the EST clones are from the same library. Since it is unusual for a syntaxin-like molecule to be without a sufficiently long stretch of hydrophobic residues at the carboxyl terminus to serve as a membrane anchor, we sought to further confirm the cDNA sequence by sequencing clones from an independent source, a human heart cDNA library (Strategene). Sequencing of three independent clones from this library revealed that they are identical to the sequences of the ESTs. The polypeptide has several potential regions that may form coiled-coil structures, as revealed by the Coils version 2.1 program (Fig. 1C). Bock and Scheller (7) have recently presented a series of ten novel syntaxinlike molecules and have numbered them consecutively as syntaxins 7 to 16 (7). The ESTs AA227632 and AA262151 corresponded to syntaxin 11 and we have thus retained the name human syntaxin 11 (hsyn11). A database search using the NCBI BLAST program revealed that the derived amino acid sequence of hsyn 11 has the highest homology with syntaxin 1, syntaxin 2, syntaxin 3 and syntaxin 4. The alignment of these sequences with hsyn11 is shown in Fig.2. Probing of a multiple tissue Northern blot revealed two transcripts of about 0.8 kb and 1.7 kb, which are particularly enriched in the heart and placenta (Fig.3). Interestingly, the transcript is of low abundance in brain such that it remained undetectable even after longer exposure of the blot. Myc-Tagged hsyn 11 Appeared to Be Cytoplasmic and Not Associated with Endomembranes All members of the syntaxin family identified to date have a stretch of hydrophobic amino acid residues at the C-terminus which serves as a membrane anchor. For hsyn11, this corresponding hydrophobic stretch does not exist. However, hsyn11 could still be tethered to a particular endomembrane through other mechanisms. To investigate this possibility, a myc epitope tag is attached to the N-terminus of hsyn11 (hsyn11-myc) and the subcellular localization of hsyn11-myc is determined by indirect immunofluorescence. As shown in Fig.4, transiently expressed hsyn11-myc in Vero (A, C and E) and COS cells (G) assumed a diffuse, cytoplasmic appearance. Spots of intense labeling that
FIG. 4. Analysis of transiently expressed myc-tagged hsyn11 (hsyn11-myc) by indirect immunofluorescence microscopy. Cells were fixed with 4% paraformyldehyde and incubated with anti-myc monoclonal antibody and rabbit polyclonal antibody against syntaxin 5 (A-B, GH) or rabbit antisera against ribophorin 1 (C, D, E and F). This is followed by incubation with FITC-labeled anti-mouse IgG and rhodaminelabeled anti-rabbit IgG. E and F is a 51 magnified view of the corresponding regions marked by * in C and D (to illustrate the difference in the morphology of the labeling). A, C, E and G - FITC channel; B, D, F and H - rhodamine channel. Note that in the case of the transfected cells in the rhodamine channels, there is a bleed through from some of the bright staining spots labeled with FITC. Bar Å 10 mm. 630
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stand out against a homogenous background probably represents cytoplasmic aggregates of the protein, not unexpected in view of the propensity of hsyn11 to form coiled-coil structures. There were no apparent structures which would suggest association of hsyn11-myc with the Golgi apparatus marked by syntaxin 5 (B) or the ER marked by ribophorin 1 (D). A magnified portion of the cytoplasm of a transfected cell clearly showed the difference between the reticular and punctate staining of the ER ribophorin 1 (F) and the homogenous labeling of hsyn11-myc (E). Finally, transient overexpression of hsyn11-myc in COS cells (G) appeared to disrupt the perinuclear labeling of the Golgi apparatus by syntaxin 5 (H). DISCUSSION The length of the sequence of hydrophobic residues which constitute the tail anchor of members of the syntaxin family typically exceeds 20 amino acids. The shortest amongst them is 17 amino acid residues in length, in the case of syntaxin 5 (6). In marked contrast, hsyn11 does not have a C-terminal hydrophobic domain of significant length. Consistent with this observation, transiently expressed hsyn11-myc appeared to be cytoplasmic and not associated with endomembranes. Beyond the syntaxins, there exist several members of the SNARE superfamily without a hydrophobic tail anchor. The neuronal SNAP25 and its more ubiquitous homolog SNAP23 are associated with membranes as a result of palmitoylation (10, 15). SNAP25 can be found in a preformed ternary complex with synaptobrevin and syntaxin in synaptic vesicles (14). Therefore, SNARE-SNARE association via coiled-coil structures may also serve to tether anchorless SNARE molecules to the membrane. Thus far, several epitope-tagged syntaxins have been faithfully localized to their respective compartments, as confirmed by subsequent availability of their respective antibodies (6, 12, 23, 27). Assuming that the myc-tag did not interfere with membrane association, the apparent cytoplasmic staining of hsyn11myc may be a result of overexpression, particularly if only a small number of membrane association sites exists. Otherwise, non-association of hsyn11-myc with endomembranes would suggests that, if hsyn11 has a role in vesicle trafficking, it functions in an nonconventional manner. ACKNOWLEDGMENT W.H. is supported by a research grant from the Institute of Molecular and Cell Biology.
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