1 Department of Physiology, Kyorin University, Mitaka, Tokyo 181-8611, Japan
2 Toyama Chemical Co. Ltd, Sinjuku, Tokyo 160-0023, Japan
*Author for correspondence (e-mail: akagawak{at}kyorin-u.ac.jp)
Accepted May 24, 2001
![]() |
SUMMARY |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: Syntaxin, Cytoplasmic domain, Transmembrane domain, Di-leucine-based motif, Intracellular localization, Antibody uptake experiment
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Many members of the mammalian syntaxin family have been identified; each localizes to specific membrane compartments along the exocytotic and endocytotic pathways. The first group of syntaxins identified (syntaxins 1A (HPC-1), 2, 3 and 4) is predominantly restricted to the plasma membrane; these syntaxins mediate constitutive and regulated vesicle transport events at the cell surface (Inoue et al., 1992; Bennett et al., 1993; Gaisano et al., 1996; Low et al., 1996). By contrast, syntaxin 18 localizes to the endoplasmic reticulum (ER) (Hatsuzawa et al., 2000), syntaxins 5, 6, 10, 11 and 16 localize to different subcompartments within the Golgi apparatus (Dascher et al., 1994; Bock et al., 1997; Watson and Pessin, 2000; Tang et al., 1998a; Valdez et al., 1999; Tang et al., 1998c) and syntaxins 7, 8, 12 and 13 are found in the post-Golgi endosomal population (Wong et al., 1998; Nakamura et al., 2000; Prekeris et al., 1999; Subramaniam et al., 2000; Tang et al., 1998b; Prekeris et al., 1998). Because of the importance of t-SNAREs in docking and fusion of transport vesicles, the roles of syntaxins in intracellular localization must be established in order to understand the mechanism of transport specificity.
The functional domains of several syntaxins have been characterized with regard to their roles in intracellular localization. First, both the transmembrane and cytoplasmic domains of Sed5p, a yeast homologue of syntaxin 5, which is required for transport of vesicles between the ER and the Golgi apparatus, have been shown to be important for localization to the cis-Golgi network (CGN) (Hardwick and Pelham, 1992; Banfield et al., 1994). Second, in mammalian cells, two isoforms of syntaxin 5 (42 kDa and 35 kDa) are believed to arise from the same gene through alternative translation initiation sites. The 42 kDa form has an N-terminal cytoplasmic extension containing a type II ER retrieval motif and localizes to the ER, whereas the 35 kDa form localizes to the CGN, as does Sed5p (Hui et al., 1997). Third, it has recently been shown that syntaxin 6 has two independent cytoplasmic regions that are responsible for its localization to the trans-Golgi network (TGN) (Watson and Pessin, 2000). One of the domains has a tyrosine-based motif that can function as a plasma membrane internalization signal (Bos et al., 1993; Humphrey et al., 1993; Banting and Ponnambalam, 1997; Bonifacino and DellAngelica, 1999). Finally, the yeast syntaxin Vam3p, which localizes to vacuoles, has a di-leucine-based motif in its cytoplasmic domain (Darsow et al., 1998). This motif is a sorting signal present in endosomal/lysosomal-targeting proteins (Darsow et al., 1998; Pond et al., 1995; Tang and Hong, 1999). However, the details of the mechanisms by which syntaxins localize to specific intracellular venues are poorly understood.
In this study, we used chimeric syntaxins to identify the syntaxin polypeptide domains that direct intracellular localization. We constructed chimeras combining the cytoplasmic and transmembrane domains of different syntaxins, and we compared their sites of intracellular localization to those of wild-type syntaxins.
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
cDNA cloning and plasmid construction
A cDNA encoding the full length of human syntaxin 1A was obtained by using the polymerase chain reaction (PCR) on total RNA isolated from the human neuroblastoma cell line NB-1. In addition, cDNAs encoding the full-length human syntaxins 5, 6, 7 and 8 were obtained by PCR of human liver and kidney cDNA libraries (Life Technologies Inc.). Although two splicing isoforms of syntaxin 5 exist, we obtained only the short (35 kDa) form, which localizes to the CGN. Primer sets were designed from sequences found in GenBank.
Chimeras were generated as follows. The cytoplasmic domain of syntaxin 1A was connected to the presumed transmembrane domain of syntaxin 5 (amino acids 280-301), 6 (amino acids 235-255), 7 (amino acids 237-261) or 8 (amino acids 215-236), resulting in syn1-5, syn1-6, syn1-7, and syn1-8, respectively. The transmembrane domain of syntaxin 1A (amino acids 266-288) was connected to the cytoplasmic domain of syntaxin 5, 6, 7 or 8, resulting in syn5-1, syn6-1, syn7-1 and syn8-1, respectively. Similarly, the transmembrane domain of syntaxin 5 was connected to the cytoplasmic domain of syntaxin 6, resulting in syn6-5.
QuickChangeTM Site-Directed Mutagenesis (Stratagene) was used to generate mutations in the putative di-leucine-based motifs found at amino acids 162-168 of syntaxin 7 and at amino acids 77-83 of syntaxin 8. The syn7-mut mutation of syntaxin 7 contained Leu167-Ile168Ala-Ala substitutions. The syn8-mut mutation of syntaxin 8 contained Leu82-Leu83
Ala-Ala substitutions.
For intracellular localization analysis, syntaxin cDNAs were subcloned into the pcDNA3-HAN vector (Shin et al., 1997) for expression in mammalian cells as fusion proteins in which the N-termini were fused to an HA tag. For antibody uptake experiments, syntaxin cDNAs were subcloned into the pcDNA3-myc3C vector for expression as fusion proteins in which the C-termini were fused to three c-myc tags.
Cell culture
PC12h cells (Watanabe et al., 1999) were cultured at 37°C in 5% CO2 in Dulbeccos modified Eagles medium containing 4 mM L-glutamine, 100 IU/ml penicillin and 100 µg/ml streptomycin, supplemented with 10% fetal calf serum and 5% horse serum. Rat Clone 9 hepatocytes were cultured at 37°C in 5% CO2 in nutrient mixture F12 medium containing 100 IU/ml penicillin and 100 µg/ml streptomycin, supplemented with 10% fetal calf serum.
Indirect immunofluorescence analysis
For electroporation, a 60-80% confluent culture of PC12h cells was trypsinized and 106 cells were resuspended with 10 µg DNA in 400 µl Dulbeccos modified Eagles medium containing 4 mM L-glutamine. After incubation on ice for 10 minutes, the cells were transferred to a Bio-Rad Gene Pulser cuvette (0.4 cm electrode gap) and electroporated with one shock at 250 mV, 960 µF, using a Bio-Rad Pulse Controller (Bio-Rad Laboratories). The cells were then incubated on ice for 10 minutes, resuspended in serum-containing medium and plated on polyethylenimine-coated Lab-Tek II chamber slides (Nunc). After incubation for 24 hours, the cells were treated with 20 mM sodium butyrate for 24 hours (for intracellular localization analysis) or for 48 hours (for antibody uptake experiments). Rat Clone 9 cells grown on eight-well Lab-Tek II chamber slides were transfected using FuGene6TM transfection reagent (Roche Diagnostics Corp.) and incubated for 12 hours (for intracellular localization analysis) or 24 hours (for intracellular localization analysis and antibody uptake experiments).
The above cells were processed for indirect immunofluorescence analysis as described previously (Torii et al., 1995). Briefly, cells were fixed and permeabilized with methanol at -20°C for 5 minutes. In some experiments, cells were fixed with 4% paraformaldehyde and then permeabilized with 0.1% Triton X-100. Where indicated, the cells were treated with 5 µg/ml BFA for 30 minutes or incubated with 8 µg/ml monoclonal mouse anti-c-myc antibody 9E10 for 3 hours before fixing. The fixed and permeabilized cells were incubated with monoclonal rat anti-HA antibody 3F10 and the indicated antibodies against organelle marker proteins, and were sequentially incubated with secondary antibodies for the intracellular localization analysis. For detection of c-myc-tagged syntaxins in antibody uptake experiments, fixed and permeabilized cells were incubated with FITC-conjugated anti-mouse IgG. The stained cells were observed with a confocal laser-scanning microscope (LSM 410 invert; Carl Zeiss).
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
In contrast to our result for syn1-5, syn1-6 localized to the plasma membrane when expressed in PC12h cells, like wild-type syntaxin 1A, although wild-type syntaxin 6 was localized to the TGN (Fig. 2c,d). Similar results were also obtained for cells expressing syn1-7 or syn1-8; in both cases, the chimeras were localized at the plasma membrane, whereas the wild-type syntaxins 7 and 8 were localized to endosomes (Fig. 2e-h). These results suggest that the cytoplasmic domains of syntaxins 6, 7 and 8 are important for their intracellular localization. Furthermore, these results suggest that the transmembrane domains of syntaxins 6, 7 and 8 are necessary for their transport to the plasma membrane but not for specific intracellular localization.
To examine further the role of the cytoplasmic domain, we generated chimeras in which the transmembrane domain of syntaxin 1A replaced the syntaxins normal transmembrane domains, resulting in syn5-1, syn6-1, syn7-1 and syn8-1. We then determined the intracellular localization of these chimeras when expressed in Clone 9 cells (Fig. 3). When syn5-1 was expressed, it colocalized with ß-COP as well as wild-type syntaxin 5. When syn6-1 was expressed, it colocalized with TGN38 as did wild-type syntaxin 6. Likewise, when syn7-1 and syn8-1 were expressed, they were localized to endosomes similar to wild-type syntaxins 7 and 8, respectively (compare Fig. 3 with Fig. 1). Similar results were also obtained in PC12h cells (data not shown). Therefore, these results indicate that both the transmembrane and cytoplasmic domains of syntaxin 5 are important for its intracellular localization, and that the cytoplasmic domains of syntaxins 6, 7 and 8 are important for their specific intracellular localization.
|
|
|
|
Di-leucine-based motifs have distinct roles in intracellular localization and trafficking of syntaxins 7 and 8
The above results suggest that the cytoplasmic domains of syntaxins 7 and 8 contain internalization signals. Di-leucine-based motifs are important sorting signals present in endosomal/lysosomal-targeting proteins and are typically composed of an acidic residue (aspartic acid or glutamic acid) followed by a pair of leucine residues (Darsow et al., 1998; Pond et al., 1995; Tang and Hong, 1999). Recently, it was shown that many syntaxins that localize to the TGN or to endosomes have putative di-leucine-based motif(s) in their cytoplasmic domains. Like these syntaxins, both syntaxins 7 and 8 contain a putative di-leucine-based motif in their cytoplasmic domains (Tang and Hong, 1999). Therefore, we examined the roles of these putative di-leucine-based motifs in syntaxins 7 and 8 by ascertaining the effects of mutations within these motifs. These wild-type and mutated motifs are shown in Fig. 7.
|
When syn8-mut was expressed at low levels in Clone 9 cells, it was localized to the perinuclear Golgi region (Fig. 7d). Moreover, in overexpressing cells, this mutant was localized only to the perinuclear Golgi region and little was found at any intracellular compartments and the plasma membrane (Fig. 7e). Antibody uptake experiments for syn8-mut-expressing cells showed labeling neither at the cell surface nor at any intracellular compartments (Fig. 7f). These results were distinctly different from those observed for wild-type syntaxin 8, suggesting that this mutated motif is normally involved in the exocytotic trafficking of syntaxin 8 from the TGN. Furthermore, these results indicate that the di-leucine-based motifs in the cytoplasmic domains of syntaxins 7 and 8 have distinct roles in intracellular localization and trafficking.
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
We determined that both the cytoplasmic and transmembrane domains of syntaxin 5 are involved in its specific intracellular localization to the CGN. These results do not conflict with results shown for the yeast homologue Sed5p (Banfield et al., 1994). By contrast, we did not find any evidence that the transmembrane domain of syntaxin 6, 7 or 8 directs specific intracellular localization of these molecules. In Fig. 2d and Fig. 5b', syn1-6 and wild-type syntaxin 6 seemed to be localized to the TGN as well as to the plasma membrane, so that the transmembrane domain of syntaxin 6 might contain a TGN-localization signal. However, if the transmembrane domain of syntaxin 6 is involved in its specific intracellular localization to the TGN, syntaxin 6 should be found only at the TGN but not at the plasma membrane even in overexpressing cells. We obtained apparent plasma membrane staining in syn1-6-expressing and wild-type syntaxin-6-expressing cells. Therefore, we considered that the transmembrane domain of syntaxin 6 has no or, if any, a weak role for its specific intracellular localization. We further demonstrated that the transmembrane domains of syntaxins 6, 7 and 8, but not of syntaxin 5, as such allow trafficking to the plasma membrane. These findings indicate that the transmembrane domain of syntaxin 5 does not permit escape from the early transport pathway. One possibility for the retention of the transmembrane domain of syntaxin 5 would be the formation of oligomeric structures with itself and/or other resident proteins of the compartment. Alternatively, it has been proposed, although not yet proven, that the length of the transmembrane domain in membrane proteins determines their fate. In support of this proposal, proteins that localize to the ER and Golgi apparatus generally possess shorter transmembrane domains than do plasma membrane proteins (Letourneur and Klausner, 1992; Rayner and Pelham, 1997). Nonetheless, we were unable to find a correlation between intracellular localization of syntaxins and the length of their transmembrane domains. Therefore, a more precise studies are necessary to estimate the role of the transmembrane domains of syntaxins.
Here, we showed that the cytoplasmic domains of syntaxins 6, 7 and 8 can direct intracellular localization and internalization from the plasma membrane. Like wild-type syntaxins 6, 7 and 8, the chimeras syn6-1, syn7-1 and syn8-1 were shown to be localized either to the TGN or to endosomes and to cycle through the plasma membrane. However, when these cytoplasmic domains were combined with the syntaxin 5 transmembrane domain, producing syn6-5, syn7-5 and syn8-5, these chimeras were retained at the CGN rather than being transported to either the TGN or the endosomes. These results again indicate that the transmembrane domain of syntaxin 5 suppresses transport at the CGN, whereas the transmembrane domains of syntaxins 6, 7 and 8 allow transport to the plasma membrane. These results further suggest that the cytoplasmic domains of syntaxins 6, 7 and 8 can function as localization signals only when these proteins are delivered, at the least, to a post-CGN compartment. It seems likely that the role of syntaxin transmembrane domains is to constrain trafficking within the transport pathway, and the role of syntaxin cytoplasmic domains is to then determine the final localization fate of syntaxins.
To identify the localization and internalization signals in the cytoplasmic domains of syntaxins 7 and 8, we examined putative di-leucine-based motifs. Di-leucine-based motifs are thought to play a role in endocytosis (Pond et al., 1995; Letourneur and Klausner, 1992) and have been found to bind to the clathrin adapter complexes AP-1, AP-2 (Heilker et al., 1996) and AP-3 (Darsow et al., 1998; Höning et al., 1998). The AP-1 adapter complex mediates the formation of clathrin-coated vesicles at the TGN and is involved in protein transport from the TGN. By contrast, the AP-2 clathrin adapter complex mediates the formation of clathrin-coated vesicles at the plasma membrane and is involved in endocytosis (Pearse and Robinson, 1990). In these experiments, we showed that the putative di-leucine-based motif of syntaxin 7 (amino acids 162-168) is involved in its internalization (Fig. 7; Fig. 8). Therefore, it seems likely that this di-leucine-based motif binds to the AP-2 clathrin adapter complex and that syntaxin 7 internalizes from the plasma membrane via a clathrin-mediated pathway.
|
As illustrated in Fig. 8, we have shown that the cytoplasmic domains of syntaxins 5, 6, 7 and 8 are important for intracellular localization. The 42 kDa syntaxin 5 isoform, which localizes to the ER, has an N-terminal cytoplasmic extension containing a type II ER retrieval motif (Hui et al., 1997). However, although the 35 kDa syntaxin 5 isoform used in this study has no identified signal motifs in its cytoplasmic domain, both the wild-type 35 kDa isoform and the syn5-1 chimera localized to the CGN. This discrepancy raises the possibility that the cytoplasmic domain of syntaxin 5 contains an unidentified intracellular localization signal for retention in the CGN. By contrast, we showed that syntaxin 8 cycles through the plasma membrane by virtue of its cytoplasmic domain. However, we could not find any internalization signal motifs in the cytoplasmic domain of syntaxin 8. A candidate for the plasma membrane internalization signal was the di-leucine-based motif at amino acids 77-83 of syntaxin 8. We showed instead that this motif is involved in trafficking syntaxin 8 from the TGN, whereas that of syntaxin 7 (amino acids 162-168) is involved in internalization. Therefore, there may be an unidentified internalization signal in the cytoplasmic domain of syntaxin 8. Alternatively, the di-leucine-based motif may function in both exocytotic and endocytotic pathways. Additional strategies for identifying signal motif(s) may be required to address these issues.
In conclusion, we have shown here that both the cytoplasmic and the transmembrane domains of syntaxins contribute to their specific localization. We suggest that TGN/endosomal syntaxins cycle through the plasma membrane. Moreover, our results indicate that the cytoplasmic domains of these syntaxins are important for their specific intracellular localization and internalization. In particular, di-leucine-based motifs appear to play distinct roles in intracellular localization and trafficking of syntaxins 7 and 8. Because many syntaxins may contain di-leucine-based motif(s) or other intracellular localization signal motif(s), further studies will enable us to understand better the mechanism of specific intracellular localization of syntaxins, as well as the specific pairing of SNARE proteins.
![]() |
ACKNOWLEDGMENTS |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Banfield, D. K., Lewis, M. J., Rabouille, C., Warren, G. and Pelham, H. G. (1994). Localization of Sed5, a putative vesicle targeting molecule, to the cis-Golgi network involves both its transmembrane and cytoplasmic domains. J. Cell Biol. 127, 357-371.[Abstract]
Banting, G. and Ponnambalam, S. (1997). TGN38 and its orthologues: roles in post-TGN vesicle formation and maintenance of TGN morphology. Biochim. Biophys. Acta 1355, 209-217.[Medline]
Bennett, M. K. and Scheller, R. H. (1993). The molecular machinery for secretion is conserved from yeast to neurons. Proc. Natl. Acad. Sci. USA 90, 2559-2563.[Abstract]
Bennett, M. K., Garcia-Arraras, J. E., Elferink, L. A., Peterson, K., Fleming, A. M., Hazuka, C. D. and Scheller, R. H. (1993). The syntaxin family of vesicular transport receptors. Cell 74, 863-873.[Medline]
Bock, J. B., Klumperman, J., Davanger, S. and Scheller, R. H. (1997). Syntaxin 6 functions in trans-Golgi network vesicle trafficking. Mol. Biol. Cell 8, 1261-1271.[Abstract]
Bonifacino, J. S. and DellAngelica, E. C. (1999). Molecular bases for the recognition of tyrosine-based sorting signals. J. Cell Biol. 145, 923-926.
Bos, K., Wraight, C. and Stanley, K. K. (1993). TGN38 is maintained in the trans-Golgi network by a tyrosine-containing motif in the cytoplasmic domain. EMBO J. 12, 2219-2228.[Abstract]
Chao, D. S., Hay, J. C., Winnick, S., Prekeris, R., Klumperman, J. and Scheller, R. H. (1999). SNARE membrane trafficking dynamics in vivo. J. Cell Biol. 144, 869-881.
Clary, D. O., Griff, I. C. and Rothman, J. E. (1990). SNAPs, a family of NSF attachment proteins involved in intracellular membrane fusion in animals and yeast. Cell 61, 709-721.[Medline]
Darsow, T., Burd, C. G. and Emr, S. D. (1998). Acidic di-leucine motif essential for AP-3-dependent sorting and restriction of the functional specificity of the Vam3p vacuolar t-SNARE. J. Cell Biol. 142, 913-922.
Dascher, C., Matteson, J. and Balch, W. E. (1994). Syntaxin 5 regulates endoplasmic reticulum to Golgi transport. J. Biol. Chem. 269, 29363-29366.
DellAngelica, E. C., Ohno, H., Ooi, C. E., Rabinovich, E., Roche, K. W. and Bonifacino, J. S. (1997a). AP-3: an adaptor-like protein complex with ubiquitous expression. EMBO J. 16, 917-928.
DellAngelica, E. C., Ooi, C. E. and Bonifacino, J. S. (1997b). ß3A-adaptin, a subunit of the adaptor-like complex AP-3. J. Biol. Chem. 272, 15078-15084.
DellAngelica, E. C., Klumperman, J., Stoorvogel, W. and Bonifacino, J. S. (1998). Association of the AP-3 adaptor complex with clathrin. Science 280, 431-434.
Fukuda, R., McNew, J. A., Weber, T., Parlati, F., Engel, T., Nickel, W., Rothman, J. E. and Söllner, T. H. (2000). Functional architecture of an intracellular membrane t-SNARE. Nature 407, 198-202.[Medline]
Gaisano, H. Y., Ghai, M., Malkus, P. N., Sheu, L., Bouquillon, A., Bennett, M. K. and Trimble, W. S. (1996). Distinct cellular locations of the syntaxin family of proteins in rat pancreatic acinar cells. Mol. Biol. Cell 7, 2019-2027.[Abstract]
Graham, T. R. and Emr, S. D. (1991). Compartmental organization of Golgi-specific protein modification and vacuolar protein sorting events defined in a yeast sec18 (NSF) mutant. J. Cell Biol. 114, 207-218.[Abstract]
Griff, I. C., Schekman, R., Rothman, J. E. and Kaiser, C. A. (1992). The yeast SEC17 gene product is functionally equivalent to mammalian alpha-SNAP protein. J. Biol. Chem. 267, 12106-12115.
Hardwick, K. G. and Pelham, H. R. (1992). SED5 encodes a 39-kD integral membrane protein required for vesicular transport between the ER and the Golgi complex. J. Cell Biol. 119, 513-521.[Abstract]
Hatsuzawa, K., Hirose, H., Tani, K., Yamamoto, A., Scheller, R. H. and Tagaya, M. (2000). Syntaxin 18, a SNAP receptor that functions in the endoplasmic reticulum, intermediate compartment, and cis-Golgi vesicle trafficking. J. Biol. Chem. 275, 13713-13720.
Heilker, R., Manning-Krieg, U., Zuber, J. F. and Spiess, M. (1996). In vitro binding of clathrin adaptors to sorting signals correlates with endocytosis and basolateral sorting. EMBO J. 15, 2893-2899.[Abstract]
Höning, S., Sandoval, I. V. and von Figura, K. (1998). A di-leucine-based motif in the cytoplasmic tail of LIMP-II and tyrosinase mediates selective binding of AP-3. EMBO J. 17, 1304-1314.
Hui, N., Nakamura, N., Sonnichsen, B., Shima, D. T., Nilsson, T. and Warren, G. (1997). An isoform of the Golgi t-SNARE, syntaxin 5, with an endoplasmic reticulum retrieval signal. Mol. Biol. Cell 8, 1777-1787.[Abstract]
Humphrey, J. S., Peters, P. J., Yuan, L. C. and Bonifacino, J. S. (1993). Localization of TGN38 to the trans-Golgi network: involvement of a cytoplasmic tyrosine-containing sequence. J. Cell Biol. 120, 1123-1135.[Abstract]
Inoue, A., Obata, K. and Akagawa, K. (1992). Cloning and sequence analysis of cDNA for a neuronal cell membrane antigen, HPC-1. J. Biol. Chem. 267, 10613-10619.
Letourneur, F. and Klausner, R. D. (1992). A novel di-leucine motif and a tyrosine-based motif independently mediate lysosomal targeting and endocytosis of CD3 chains. Cell 69, 1143-1157.[Medline]
Lippincott-Schwartz, J., Yuan, L. C., Bonifacino, J. S. and Klausner, R. D. (1989). Rapid redistribution of Golgi proteins into the ER in cells treated with brefeldin A: evidence for membrane cycling from Golgi to ER. Cell 56, 801-813.[Medline]
Low, S. H., Chapin, S. J., Weimbs, T., Komuves, L. G., Bennett, M. K. and Mostov, K. E. (1996). Differential localization of syntaxin isoforms in polarized Madin-Darby canine kidney cells. Mol. Biol. Cell 7, 2007-2018.[Abstract]
McNew, J. A., Parlati, F., Fukuda, R., Johnston, R. J., Paz, K., Paumet, F., Söllner, T. H. and Rothman, J. E. (2000). Compartmental specificity of cellular membrane fusion encoded in SNARE proteins. Nature 407, 153-159.[Medline]
Nakamura, N., Yamamoto, A., Wada, Y. and Futai, M. (2000). Syntaxin 7 mediates endocytic trafficking to late endosomes. J. Biol. Chem. 275, 6523-6529.
Newman, L. S., McKeever, M. O., Okano, H. J. and Darnell, R. B. (1995). ß-NAP, a cerebellar degeneration antigen, is a neuron-specific vesicle coat protein. Cell 82, 773-783.[Medline]
Palade, G. (1975). Intracellular aspects of the process of protein synthesis. Science 189, 347-358[Medline]
Parlati, F., McNew, J. A., Fukuda, R., Miller, R., Söllner, T. H. and Rothman, J. E. (2000). Topological restriction of SNARE-dependent membrane fusion. Nature 407, 194-198.[Medline]
Pearse, B. M. and Robinson, M. S. (1990). Clathrin, adaptors, and sorting. Annu. Rev. Cell Biol. 6, 151-171.
Pond, L., Kuhn, L. A., Teyton, L., Schutze, M. P., Tainer, J. A., Jackson, M. R. and Peterson, P. A. (1995). A role for acidic residues in di-leucine motif-based targeting to the endocytic pathway. J. Biol. Chem. 270, 19989-19997.
Prekeris, R., Klumperman, J., Chen, Y. A. and Scheller, R. H. (1998). Syntaxin 13 mediates cycling of plasma membrane proteins via tubulovesicular recycling endosomes. J. Cell Biol. 143, 957-971.
Prekeris, R., Yang, B., Oorschot, V., Klumperman, J. and Scheller, R. H. (1999). Differential roles of syntaxin 7 and syntaxin 8 in endosomal trafficking. Mol. Biol. Cell 10, 3891-3908.
Pryer, N. K., Wuestehube, L. J. and Schekman, R. (1992). Vesicle-mediated protein sorting. Annu. Rev. Biochem. 61, 471-516.[Medline]
Rayner, J. C. and Pelham, H. R. (1997). Transmembrane domain-dependent sorting of proteins to the ER and plasma membrane in yeast. EMBO J. 16, 1832-1841.[Abstract]
Reaves, B. and Banting, G. (1992). Perturbation of the morphology of the trans-Golgi network following Brefeldin A treatment: redistribution of a TGN-specific integral membrane protein, TGN38. J. Cell Biol. 116, 85-94.[Abstract]
Rothman, J. E. and Warren, G. (1994). Implications of the SNARE hypothesis for intracellular membrane topology and dynamics. Curr. Biol. 4, 220-233.[Medline]
Rothman, J. E. and Wieland, F. T. (1996). Protein sorting by transport vesicles. Science 272, 227-234.[Abstract]
Schekman, R. and Orci, L. (1996). Coat proteins and vesicle budding. Science 271, 1526-1533.[Abstract]
Söllner, T., Whiteheart, S. W., Brunner, M., Erdjument-Bromage, H., Geromanos, S., Tempst, P. and Rothman, J. E. (1993a). SNAP receptors implicated in vesicle targeting and fusion. Nature 362, 318-324.[Medline]
Söllner, T., Bennett, M. K., Whiteheart, S. W., Scheller, R. H. and Rothman, J. E. (1993b). A protein assembly-disassembly pathway in vitro that may correspond to sequential steps of synaptic vesicle docking, activation, and fusion. Cell 75, 409-418.[Medline]
Shin, H. W., Shinotsuka, C., Torii, S., Murakami, K. and Nakayama, K. (1997). Identification and subcellular localization of a novel mammalian dynamin-related protein homologous to yeast Vps1p and Dnm1p. J. Biochem. (Tokyo) 122, 525-530.[Abstract]
Simpson, F., Peden, A. A., Christopoulou, L. and Robinson, M. S. (1997). Characterization of the adaptor-related protein complex, AP-3. J. Cell Biol. 137, 835-845.
Subramaniam, V. N., Loh, E., Horstmann, H., Habermann, A., Xu, Y., Coe, J., Griffiths, G. and Hong, W. (2000). Preferential association of syntaxin 8 with the early endosome. J. Cell Sci. 113, 997-1008.
Tang, B. L. and Hong, W. (1999). A possible role of di-leucine-based motifs in targeting and sorting of the syntaxin family of proteins. FEBS Lett. 446, 211-212.[Medline]
Tang, B. L., Low, D. Y., Lee, S. S., Tan, A. E. and Hong, W. (1998a). Molecular cloning and localization of human syntaxin 16, a member of the syntaxin family of SNARE proteins. Biochem. Biophys. Res. Commun. 242, 673-679.[Medline]
Tang, B. L., Tan, A. E., Lim, L. K., Lee, S. S., Low, D. Y. and Hong, W. (1998b). Syntaxin 12, a member of the syntaxin family localized to the endosome. J. Biol. Chem. 273, 6944-6950.
Tang, B. L., Low, D. Y., Tan, A. E. and Hong, W. (1998c). Syntaxin 10: a member of the syntaxin family localized to the trans-Golgi network. Biochem. Biophys. Res. Commun. 242, 345-350.[Medline]
Torii, S., Banno, T., Watanabe, T., Ikehara, Y., Murakami, K. and Nakayama, K. (1995). Cytotoxicity of brefeldin A correlates with its inhibitory effect on membrane binding of COP coat proteins. J. Biol. Chem. 270, 11574-11580.
Valdez, A. C., Cabaniols, J. P., Brown, M. J. and Roche, P. A. (1999). Syntaxin 11 is associated with SNAP-23 on late endosomes and the trans-Golgi network. J. Cell Sci. 112, 845-854.
Watanabe, T., Fujiwara, T., Komazaki, S., Yamaguchi, K., Tajima, O. and Akagawa, K. (1999). HPC-1/syntaxin 1A suppresses exocytosis of PC12 cells. J. Biochem. (Tokyo) 125, 685-689.[Abstract]
Watson, R. T. and Pessin, J. E. (2000). Functional cooperation of two independent targeting domains in syntaxin 6 is required for its efficient localization in the trans-golgi network of 3T3L1 adipocytes. J. Biol. Chem. 275, 1261-1268.
Wong, S. H., Xu, Y., Zhang, T. and Hong, W. (1998). Syntaxin 7, a novel syntaxin member associated with the early endosomal compartment. J. Biol. Chem. 273, 375-380.