The syntaxin family of vesicular transport receptors

Cell, Vol. 74, 663-673,

September

10, 1993, Copynght

0 1993 by Cell Press

The Syntaxin Family of Vesicular Transport Receptors

Mark K. Bennett, Jose E. Garcia-Arraras,’ Lisa A. Elferink, Karen Peterson, Anne M. Fleming, Christopher D. Hazuka, and Richard H. Scheller Department of Molecular and Cellular Physiology Howard Hughes Medical Institute Stanford University Medical Center Stanford, California 94305

Summary Syntaxins A and B are nervous system-specific proteins implicated in the docking of synaptic vesicles with the presynaptic plasma membrane. A family of syntaxin-related proteins from rat has been identified that shares 23%-84% amino acid identity. Each of the six syntaxins terminate with a carboxy-terminal hydrophobic domain that anchors the protein on the cytoplasmic surface of cellular membranes. The syntaxins display a broad tissue distribution and, when expressed in COS cells, are targeted to different subcellular compartments. Microinjection studies suggest that the nervous system-specific syntaxin 1A is important for calcium-regulated secretion from neuroendocrine PC12 cells. These results indicate that the syntaxins are a family of receptors for intracellular transport vesicles and that each target membrane may be identified by a specific member of the syntaxin family. Introduction Neurons communicate with their target cells via synaptic transmission. One of the central elements in this process is the regulated release of neurotransmitter from the presynaptic nerve terminal. This release is accomplished by the calcium-triggered fusion of neurotransmitter-containing synaptic vesicles with the presynaptic plasma membrane at a specialized region called the active zone. Biochemical dissection of the process of neurotransmitter release has focused on the identification of proteins that are localized to the synaptic vesicle or the presynaptic active zone. Through the characterization of these synaptic proteins, an understanding of various aspects of membrane trafficking in the nerve terminal is emerging (for review see Kelly, 1993). In addition, because the synaptic vesicle life cycle includes the general cellular processes of protein sorting, vesicle targeting, membrane fusion, and endocytic membrane recycling, its characterization is providing insight into the mechanisms that underlie vesiclemediated membrane transport along the constitutive secretory and endocytic pathways in all eukaryotic cells (Bennett and Scheller, 1993). Two of the central steps in the synaptic vesicle life cycle ‘Present address: Biology Department, Piedas, Puerto Rico 00931-3360.

University of Puerto Rico, Rio

are the docking of vesicles with the presynaptic plasma membrane and the subsequent fusion of these membranes in response to calcium. We have identified a nervous system-specific protein, syntaxin, that is localized to the plasma membrane and interacts with both the synaptic vesicle protein synaptotagmin (~65) and a class of calcium channels (N-type) that is involved in the regulation of neurotransmitter release (Bennett et al., 1992; lnoue et al., 1992; Morita et al., 1992). Based on these observations, we proposed that syntaxin may be involved in the docking or fusion (or both) of synaptic vesicles with the plasma membrane. Recently, three yeast genes (SED5, PEP72, and SSO7) have been identified that encode proteins displaying sequence homology to syntaxin, each of which plays a role in the secretory pathway. The SfD5 gene, identified by its ability to suppress the loss of the endoplasmic reticulum (ER) retention receptor erd2, is required for efficient membrane transport between the ER and the Golgi complex (Hardwick and Pelham, 1992). The PEP72 gene is required for efficient delivery of proteolytic enzymes from the Golgi complex to the vacuole (Jones, 1976; K. A. Becherer and E. W. Jones, personal communication), and the SSO7 gene product acts as a suppressor of the late-acting (post-Golgi) secretory mutant SEC7 (S. Keranen, personal communication). The fact that different syntaxin-like proteins are required at three stages of the yeast secretory pathway suggests that these proteins may define the specificity of each vesicular trafficking step (Bennett and Scheller, 1993). Given the proposed role of syntaxin in synaptic vesicle docking with the plasma membrane, it is possible that the syntaxin-related proteins function as target membrane-specific receptors for transport vesicle docking, fusion, or both. The recent identification of syntaxin as one of several membrane proteins capable of forming a complex with the N-ethylmaleimide-sensitive fusion protein (NSF) and the soluble NSF attachment protein (SNAP), soluble factors required for transport vesicle docking and fusion at multiple stages of the secretory and endocytic pathways (SolIner et al., 1993) further supports this hypothesis. In the current study, we describe a family of mammalian syntaxin-related proteins that are expressed with broad tissue distribution and are localized to different membrane compartments. In addition, we demonstrate that syntaxin 1A is cytoplasmically oriented and that soluble syntaxin 1A fragments or an antibody generated against syntaxin 1A is able to disrupt calcium-regulated secretion from neuroendocrine PC12 cells. These results suggest that syntaxins play a general role in membrane trafficking, including regulated secretion, by acting as target membrane-specific receptors for transport vesicles. Results A Family of Syntaxin Proteins in Rat To identify additional members of the syntaxin family, a variety of rat cDNA libraries were screened at low strin-

Cell 864

Frgure 1 The Rat Syntaxin

body capable of blocking epithelial cell morphogenesis (Hirai et al., 1992). The relationship between epimorphin and membrane trafficking is unclear (Pelham, 1993; Hirai et al., 1993; see Discussion). When the sequences of syntaxins l-4 were compared with the yeast syntaxin-related proteins (Table l), the highest level of homology was observed between syntaxin lA/l B and the SSOl gene product (270/o-29% identity), a yeast protein likely to be involved in Golgi complex to plasma membrane transport. To identify a rat homolog of the yeast SED5 gene product, a protein implicated in ER to Golgi membrane transport, a rat macrophage cDNA library was screened at low stringency with a probe derived from a Drosophila homolog of SED5 (T. Schiipbach and I. Dawson, personal communication). A single cDNA clone was isolated that encodes a candidate rat SED5 homolog that we refer to as syntaxin 5 (Figure 1; Table 1). While syntaxin 5 displays only limited homology to the other five mammalian syntaxin sequences (230/o-26% identity), it is most similar to yeast SED5 (35% identity). Similarly, the SED5 gene product is more homologous to syntaxin 5 than to the products of the yeast genes PEP72 and SSO7, suggesting that SED5 and syntaxin 5 may be functional homologs. In addition to sharing varying levels of sequence homology, the mammalian syntaxin proteins also exhibit several common structural features. All are 288-301 amino acids in length and end with a region of highly hydrophobic amino acids at the extreme carboxyl terminus (boxed in Figure 1). This domain isof sufficient length and hydrophobicity to serve as a membrane anchor. Syntaxin lA/lB behaves as an integral membrane protein (Bennett et al., 1992). In addition, expression of full-length and truncated syntaxin 1A in COS cells demonstrated that the carboxyterminal hydrophobic domain is necessary for membrane attachment (data not shown). Each member of the syntaxin family also contains several domains predicted to form a-helical coiled-coil structures (Inoue et al., 1992; Spring et al., 1993) regions likely to be involved in proteinprotein interactions. One such coiled-coil region near the carboxy-terminal hydrophobic domain is within a 70 amino acid segment that displays the highest level of sequence homology both within the rat syntaxin family (Figure 1) and between rat and yeast proteins (Pelham, 1993). Biochemi-

Family

Alignment of the ammo acid sequences predicted from six rat syntaxin (Syn) cDNA clones, Optimal alignment was produced with the Pile-Up program. Residues that are stippled are identical in at least four of the syntaxin rsoforms, and the boxed region indicates the carboxy-terminal hydrophobic domain. In two independent isolates of syntaxrn 2, the sequence TQTLSPPGR was inserted between amino acids 225 and 226.

gency with probes derived from the syntaxin cDNA clones previously isolated from rat brain (syntaxins A and 6; Bennett et al., 1992). Three additional cDNA clones were isolated that are highly homologous to syntaxins A and B. Figure 1 illustrates the alignment of the predicted amino acid sequences derived from the five syntaxin cDNA clones. Owing to their high level of sequence homology (84% identity; Table l), syntaxins A and B have been renamed syntaxins 1A and lB, while the three additional syntaxin isoforms have been named syntaxins 2-4. The sequence similarity observed between syntaxins 2-4 and syntaxin lA/l B ranges from 460/o-64% identity (Table 1). Whereas syntaxins 3 and 4 represent novel protein sequences, syntaxin 2 shares 96% amino acid identity with mouse epimorphin (suggesting it is the rat homolog of this protein). The cDNA clone encoding epimorphin was isolated in an expression screen using a monoclonal anti-

Table 1. Syntaxin Family Sequence synlA synlA synlB syn2 syn3 syn4 syn5 SSOl SED5 PEP12

91 79 77 65 46 53 45 48

synlB 84(t) 82 77 67 45 53 48 51

Homologies syn2 63(l) 64(t) -

syn3

syn4

syn5

SSOl

SED5

PEP12

W3)

‘W) ‘WY

2V)

2W

W-Y WW

2W)

79 67 44 51 50 47

62 46 50 47 50

61(3)

W3)

44(5) 41(5) 45 52 45 51

23(8) 21(5) 22(7)

2W 49 57 51

2W)

2363 2W 2W 2W) -

46 52

22(10)

2W)

2W 2W) 2.W)

35(5)

20(7)

2’39) 21(g)

21(6)

216%

50

2wJ) -

The percent amino acid identity and similarity between rat and yeast syntaxin isoforms are displayed. Values above the diagonal represent percent amino acid identity, with the number of gaps introduced to produce optimal alignment presented in parentheses. Values below the diagonal represent percent amino acid similarity with the following conservative changes: D-E, R-K, S-T, N-Q, and I-L-M-V. Optimal alignments were produced by the FASTA program.

A Family of Syntaxin-Related 865

KYOSKARR2L\

Figure 2. Alternative

/$

Proteins

KKjWIIAAVVVAVIAVLALIIGLSVqK MFVLlCVVTLLVlLGIILATALS\

A

a-factor

syntaxin

A

5670

1234

GVLCALGROC

Forms of Syntaxin 2

Three alternative carboxy-terminal sequences of syntaxin 2 (syn2) diverge after amino acid 264. Boxed sequences represent the hydrophobic domains of syntaxins 2 and 2’.

cal evidence suggests that syntaxin lA/l B interacts with a number of other proteins, including synaptotagmin, N-type calcium channels, and an NSF-SNAP complex (Bennett et al., 1992; Morita et al., 1992; Sbllner et al., 1993). The conserved coiled-coil domains may be responsible for these or other interactions. The analysis of multiple cDNA clones encoding syntaxin 2 revealed variability in the carboxy-terminal domain. In addition to the hydrophobic membrane anchor that is highly similar to mouse epimorphin, two alternative carboxy-terminal sequences were identified (Figure 2). While in one isoform (syntaxin 2’) the carboxy-terminal sequence was replaced with one of equal length and hydrophobicity, in the other (syntaxin 2”) it was replaced by a 10 amino acid sequence including only six hydrophobic residues (hydropathy index, >1.8; Kyte and Doolittle, 1982). Although this domain is not of sufficient length or hydrophobicity to span the membrane, it does contain two cysteine residues that could be potential sites for the posttranslational attachment of lipids. Localization studies suggested that at least a portion of syntaxin 2” is membrane associated (data not shown). It is likely that alternative splicing accounts for the variation in syntaxin 2 sequences since the carboxy-terminal sequences diverge at precisely the same site (after amino acid 264) with identical sequences preceding this site. Further experiments will be required to determine the origin and functional significance of the alternative forms of syntaxin 2. No evidence of alternative splicing was found in the analysis of multiple independent isolates of the other mammalian syntaxin isoforms. Syntaxins Are Oriented toward the Cytoplasm Since none of the syntaxin sequences include an aminoterminal signal sequence, one would expect this family of proteinsto becytoplasmicallyoriented. However, previous reports suggest that epimorphin (syntaxin 2) and syntaxin 1A (HPC-1) are oriented extracellularly (Hirai et al., 1992; lnoue et al., 1992). To resolve this issue, the membrane topology of syntaxin 1A was investigated by both in vitro translation and immunofluorescence microscopy. In vitro translation of proteins in the presence of pancreatic microsomes can be used to assess membrane topology (Figure 3A). For example, the secreted protein a factor is both core glycosylated and protected from proteolysis following translation in the presence of microsomes. Conversely, when syntaxin 1 A messenger RNA (mRNA) is translated in the presence of microsomes, the translation product is neither core glycosylated, in spite of three potential N-linked glycosylation sites, nor protected from proteoly-

gT:z

43 29 -

f

18-+

+

LII

B

Figure 3. Syntaxins

Are Cytoplasmically

Oriented

(A) In vitro translation of a factor (left) and syntaxin IA transcripts (right) was performed in a rabbit reticulocyte lysate either in the absence (lanes 1 and 5) or presence (lanes 2-4 and 6-8) of pancreatic microsomes. The [35S]methionine-labeled translation products were either left untreated (lanes 1, 2, 5, and 6) or treated with proteinase K, either in the absence (lanes 3 and 7) or presence (lanes 4 and 8) of Triton X-100, prior to analysis by SDS-polyacrylamide gel electrophoresis and autoradiography. The positions of the precursor and core-glycosylated a factor as well as the syntaxin 1A translation product are indicated by arrows. The positions of molecular weight standards are indicated at the left. (B) Indirect immunofluorescence localization of endogenous syntaxin 1A with an anti-syntaxin polyclonal antibody in permeabilized (top panel) or intact (bottom panel) PC12 cells. Scale bar, 36 Km.

sis. Fractionation studies demonstrated that the in vitro translated syntaxin 1A is associated with the microsomes (data not shown). These results support the prediction that syntaxin 1A is membrane anchored and cytoplasmically oriented. Further evidence for the cytoplasmic orientation of syntaxin was obtained by immunofluorescence localization studies in PC12 cells, a neuroendocrine cell line. PC12 cells express several synaptic vesicle proteins (CliftO ’Grady et al., 1990) as well as syntaxin 1 A, as determined by Northern blot analysis (data not shown). Using a polyclonal antibody generated against bacterially expressed syntaxin 1 A, we previously demonstrated that

Cell 866

kb

12345678

1A 27-

1B 4.5 -

4 2.520-

ii

c J

2.5 -

Figure 4 Syntaxrns Are Broadly and Drfferentially

Expressed

Nylon membranes displaying poly(A)’ RNA from eight different rat tissues were hybridized wrth 3ZP-labeled probes derived from the six drfferent syntaxin isoforms (iA, 16, and 2-5) as well as f3-actin (act) as described rn Experimental Procedures. RNA from the following tissues was analyzed: heart (lane 1). brain (lane 2) spleen (lane 3). lung (lane 4), liver (lane 5) skeletal muscle (lane 6) kidney (lane 7) and testis (lane 8). The sizes (in kilobases) of the major transcripts are Indicated. A 0.9 kb transcript was variably detected with the syntaxm 1 S probe (data not shown)

syntaxin immunoreactivity was primarily localized to the plasma membrane of synaptic vericosities in dissociated rat hippocampal cultures (Bennett et al., 1992). When the anti-syntaxin antibody was used to label PC12 cells, syntaxin immunoreactivity was primarily localized on the plasma membrane, although with a nonuniform staining pattern (Figure 3B, top panel). When PC12 cells were labeled with the antibody prior to fixation and permeabilizabon, no immunoreactivity was detected (Figure 3B, bottom panel). This indicates that syntaxin 1A is localized to the cytoplasmic surface of the plasma membrane, suggesting that previous reports describing an extracellular orientation for syntaxins 1A and 2 are incorrect. Given the structural similarities observed among the different syntaxin isoforms, it is likely that they will all have the membrane topology demonstrated here for syntaxin 1 A.

The Syntaxin Family Is Broadly Expressed Syntaxins 1A and 1 B were isolated from brain and proposed to function in synaptic vesicle docking. If syntaxin isoforms function in more general membrane trafficking pathways, they might be broadly expressed in many tissues. To determine the tissue distribution of syntaxin isoform expression, Northern blot analysis was performed on RNA isolated from eight different rat tissues (Figure 4). Whereas transcripts for syntaxins 1 A and 1 B were expressed exclusively in brain, transcripts for syntaxins 2 and 5, as well as the control j3-actin transcripts, were expressed in all tissues analyzed. Multiply sized transcripts of syntaxin 2 were expressed in liver and testis. Whether this relates to alternative spliced forms of syntaxin 2 remains to be determined. The transcript for syntaxin 3 was abundant in heart, spleen, lung, and kidney, whilesyntaxin 4 was expressed predominantly in heart, spleen, skeletal muscle, and kidney. The expression of syntaxin isoforms in a wide variety of tissues supports the hypothesis that this family of proteins is involved in the regulation of multiple membrane trafficking pathways. The ubiquitous expression of syntaxins 2 and 5 suggests an involvement in pathways that are common to all cell types, while the more restricted pattern of expression for other syntaxin isoforms (syntaxins lA/lB, 3, and 4) suggests an involvement in more specialized membrane trafficking pathways (i.e., neurotransmitter release for syntaxin 1 A/l B). Interestingly, another family of proteins proposed to be involved in the regulation of membrane trafficking, the flab family of low molecular weight GTP-binding proteins, also displays a broad yet differential expression pattern (Elferink et al., 1992). However, the restricted expression patterns of syntaxins 3 and 4 do not correlate with those of any of the known flab proteins. This suggests that distinct membrane trafficking pathways may use different combinations of these two gene families for their regulation. Syntaxins Are Localized to Distinct Subcellular Compartments If the different syntaxin isoforms are involved in the regulation of specific membrane trafficking steps, these proteins should reside on different membrane compartments. To test this possibility, five syntaxin isoforms (1 A, 2, 2”, 4, and 5) were epitope tagged on their amino termini and transiently expressed in COS cells, and their intracellular localizations were determined by indirect immunofluorescence microscopy. The epitope used in these studies was a 9 amino acid sequence from influenza hemagglutinin (HA) (Field et al., 1988). Four of the five syntaxins tested were localized predominantly on the plasma membrane. Figure 5A illustrates the colocalization of epitope-tagged syntaxin 2 (top panel) with the plasma membrane, as visualized by nonpermeabilized staining with rhodamineconjugated concanavalin A (bottom panel). The staining pattern was reminiscent of that observed for wild-type (non-epitope-tagged) syntaxin 1A in transfected COS cells, indicating that the HA epitope does not interfere with syntaxin membrane targeting. Similar results were obtained with epitope-tagged syntaxins 1A, 2”, and 4 (data

A Family of Syntaxin-Related 667

Figure 5. Syntaxins

Proteins

Are Localized

to Different Intracellular

Compartments

Indirect immunofluorescence localization of syntaxin isoforms was performed following transient transfection of COS cells as described in Experimental Procedures. (A) Double fluorescence localization of epitope-tagged syntaxin 2 (top panel) and surface concanavalin A-binding sites (bottom panel). (B) Double immunofluorescence localization of epitope-tagged syntaxin 5 (top panel) and b.COP (bottom panel). Scale bar, 24 Km.

not shown). The plasma membrane localization of syntaxin 1A is consistent with its distribution in hippocampal neurons (Bennett et al., 1992) and PC12 cells (see Figure 3B). The fact that epitope-tagged syntaxin 2”, which lacks a transmembrane anchor, was also detected on the plasma membrane suggests that it either associates with another membrane protein or that it is posttranslationally modified by a lipid moiety. Although each of these syntaxins was detected primarily on the plasma membrane, variable amounts of intracellular staining were also observed. The additional staining often appeared as a perinuclear or reticular pattern reminiscent of the Golgi complex or the ER, respectively. This may be due to either the high levels of expression produced with this system or the mislocalization of rat syntaxins in a monkey cell line. In contrast with the other syntaxins, epitope-tagged syntaxin 5 was not detected on the plasma membrane even at high expression levels, but rather was exclusively localized to intracellular membranes. The compartment to which syntaxin 5 is localized (Figure 56, top panel) displays a perinuclear distribution that extensively but not completely overlaps with B-COP (Figure 5B, bottom panel), a Golgi marker predominantly localized to the cis side of the Golgi complex (Orpins et al., 1993). In addition, syntaxin 5 staining was largely distinct from mannose8-

phosphate receptor-containing late endosomes (data not shown). The yeast SfD5 gene product is also localized to a Set of cytoplasmic vesicles that appear to be distinct from the ER and only partially overlapping with the Golgi complex (Hardwick and Pelham, 1992). Further experiments will be required to determine whether syntaxin 5 is targeted to a bona fide Golgi compartment or to the ERGolgi intermediate compartment, a tubulovesicular membrane structure that has been proposed to be the fusion target of E&derived transport vesicles and the site from which resident ER proteins are recycled (LippincottSchwartz et al., 1990; Hauri and Schweizer, 1992). The membrane topology and differential localization of the syntaxin isoforms are consistent with the possibility that different syntaxins may serve as receptors for the docking of transport vesicles at multiple stages of ihe secretory pathway. A Role for Syntaxin 1A in Calcium-Regulated Secretion from PC12 Cells To address the possibility that syntaxins function in transport vesicle docking or fusion, the role of syntaxin 1A in regulated secretion from PC12 cells was investigated. PC12 cells express a membrane-associated form of the enzyme dopamine B-hydroxylase (DPH) on the lumenal

Cell 868

A Family of Syntaxin-Related 869

Proteins

Figure 7. Soluble Syntaxins and Anti-Syntaxrn cretion from Microinjected PC12 Cells

Antibody

Reduce Se-

PC12 cells were microinjected and processed as described in the legend to Figure 6. In (A), cells were microinjected with a control preparation from bacteria expressing GST only, syntaxin fragment 11, or syntaxin fragment 16. In (B), cells were microinjected with Texas redconjugated dextran (Texas red), control F(ab’),, or anti-syntaxin polyclonal Fab (pFab). The number of fluorescent DbH patches detected on the surface of microinjected cells was expressed as a percentage of the total number of cells microinjected. Data are shown as the mean f SD from at least three separate experiments. The total number of injected cells were as follows: GST only, 372; syntaxin 11, 381; syntaxin 16, 315; Texas red, 275; F(ab’),, 421; and syntaxin pFab, 419. Asterisks indicate bins for which highly significant differences (p Q 0.01) were observed between the experimental and control preparations (calculated using an unpaired Student’s t test).

side of catecholamine-containing granules. When the cells are depolarized in the presence of calcium, granule fusion with the plasma membrane results in the exposure of Df3H on the cell surface, where it can be quantitatively detected by immunofluorescence microscopy (Elferink et al., 1993). Utilizing this assay, we have analyzed the effects that microinjection of two soluble fragments of syntaxin 1A or antibodies generated against syntaxin 1A have on calcium-regulated secretion. Soluble fragments of syntaxin 1A were expressed in bacteria as glutathione S-transferase (GST) fusion proteins and purified by glutathioneagarose chromatography and thrombin cleavage (Guan and Dixon, 1991). One soluble syntaxin fragment included

Figure 8. Syntaxin

1A Is Involved in Regulated

the entire cytoplasmic domain, lacking only the transmembrane anchor (syntaxin 11, amino acids 4-267). The other fragment corresponded to the region adjacent to the transmembrane domain that displays the highest level of sequence conservation among syntaxin isoforms (syntaxin 16, amino acids 194-267). Microinjection of syntaxin 16 efficiently reduced the appearance of Df3H staining on the cell surface (Figure 6B). Cells microinjected with a control preparation derived from bacteria expressing GST without syntaxin sequences (GST only) displayed levels of Df3H surface staining comparable to uninjected cells (Figure 6A). Quantitation of the secretion assay, obtained by counting the number of patches of D6H staining on the cell surface, is presented in Figure 7. High levels of DbH surface staining were observed in cells microinjected with either Texas red-conjugated dextran (Figure 78) GST only (Figure 7A), or a nonspecific F(ab’)n fragment (Figure 78). Previous studies also demonstrated that a variety of control or antibody preparations do not affect the levels of Df3H staining (Elferink et al., 1993). Microinjection of the two soluble fragments of syntaxin (11 and 16) each caused a significant decrease in the percentage of cells with high levels of surface D6H and a corresponding increase in the percentage of cells with low levels of surface Df3H (Figure 7A). Similar results were obtained with cells injected with Fab fragments derived from an affinitypurified anti-syntaxin 1A polyclonal antiserum (Figure 78). Although less dramatic, microinjection of a monoclonal antibody against syntaxin (HPC-1; lnoue et al., 1992) was also able to reduce surface D6H staining (data not shown). These results indicate that syntaxin 1A is an important component of the regulated secretory machinery in PC1 2 cells. Discussion During intracellular membrane trafficking, transport vesicles must recognize and fuse with the appropriate target compartment. This specificity is required to maintain the physical and functional compartmentalization of the secretory and endocytic pathways. It is likely that this transport fidelity is accomplished by specific so-called address proteins that are localized to the vesicle surface when it buds from the donor compartment and that are subsequently recognized by receptor proteins present on the target membrane (Bennett and Scheller, 1993; SolIner et al., 1993). Our results describing the localization of syntaxin isoforms to distinct membrane compartments, their broad tissue distribution, and the biochemical and functional properties of syntaxin 1 A strongly support the possibility

Secretion

Double immunofluorescence micrographs illustrating the effects of microinjection on the evoked appearance of DbH on the surface of PC12 cells. Cells were coinjected with Texas red-conjugated dextran and either soluble syntaxin fragment 16 (8) or a GST fusion protein control (A). One hour following microinjection, the cells were K’ depolarized in the presence of calcium for 10 min at 37%, fixed, and processed for surface Dj3H immunoreactivity (visualized with a fluorescein-conjugated anti-rabbit IgG secondary antibody). Large arrows indicate injected cells, and small arrows designate uninjected cells. Scale bar, 15 pm.

Cell 870

that this family of proteins acts as target membranespecific receptors for transport vesicles. The localization of syntaxin 5, for example, to the Golgi complex or an ERGolgi intermediate compartment is consistent with a role for this protein in ER-derived transport vesicle docking and further suggests functional homology with the yeast SEDS gene product. Biochemical and functional evidence indicate that syntaxin lA/l B is involved in the docking and/or fusion reaction between neurotransmitter-containing vesicles and the plasma membrane. The localization of syntaxin lA/l B to the plasma membrane, therefore, further supports the hypothesis that syntaxins serve as transport vesicle receptors At least two other syntaxins (2 and 4) are also localized to the plasma membrane. This may reflect the fact that the plasma membrane is the fusion partner for several different classes of transport vesicles. Constitutive secretory vesicles derived from the trans-Golgi network as well as endocytic recycling vesicles are destined to fuse with the plasma membrane. In addition, several different classes of regulated secretory vesicles exist in a variety of cell types, each of which, with the appropriate stimulation, fuses with the plasma membrane. Furthercompartmentalzation is presented by polarized epithelial cells that maintain two different plasma membrane domains, each of which is likely to be the fusion partner for distinct constitutive secretory, recycling endocytic, and transcytotic transport vesicles. Further studies will be required to establish whether syntaxins 2-4 (or other syntaxins) might be involved in these diverse plasma membrane fusion reactions. Since the primary screen for syntaxin isoforms utilized probes derived from syntaxin lA/l B, these experiments may have specifically selected proteins involved in the later stages of the secretory pathway. The localization of three of the syntaxins to the plasma membrane, sharing as little as44% amino acid identity, supports this possibility. It is likely, therefore, that the six syntaxin isoforms identified here are representative members of a larger multigene family, the full diversity of which remains to be determined. Although structurally conserved, the syntaxin family of proteins does not share common sequence motifs such as those found in other multigene families. For example, the level of sequence similarity among the three yeast syntaxin-related proteins, genetically shown to be required at distinct stages of the secretory pathway, is quite low (21%-230/o identity). Similarly, the homology between syntaxin 1 A/l B and syntaxin 5, representing the extremes of the secretory pathway in rat, is low (23% identity). This low overall sequence similarity among syntaxins functioning at different stages of the secretory pathway may make it difficult to identify a Golgi-specific or endosome-specific syntaxin by molecular genetic techniques alone. One of the syntaxin isoforms, syntaxin 2, is nearly identical to mouse epimorphin. Epimorphin was originally identified as a 150 kd antigen recognized by a monoclonal antibody capable of blocking the differentiation of hair follicles induced by mesenchymal cells (Hirai et al., 1992). However, when the antibody was used to screen an expression library, a single positive cDNA clone was identified that

encodes a 34 kd protein referred to as epimorphin. The results of the present and previous studies (Hardwick and Pelham, 1992; Pelham, 1993) raise questions concerning the size, topology, and function of epimorphin. The expression of alternative forms of syntaxin 2 that we have observed is not sufficient to account for the size and topology differences with epimorphin. The fact that alternative syntaxin 2 carboxyl termini are generated by alternative splicing suggests that this portion of the molecule serves a specific function beyond simply anchoring the protein in the membrane. Perhaps the hydrophobic carboxyl terminus is involved in localization to specific domains of the plasma membrane or participates in the actual fusion event. What are the proteins on the transport vesicle that act as address markers in defining fusion specificity? One possibility is the well-characterized Rab family of low molecular weight GTP-binding proteins. This family, which includes rab3A localized to the synaptic vesicle (Fischer von Mollard et al., 1990) has been implicated in the regulation of a variety of intracellular membrane trafficking events (Chavrier et al., 1990; Gorvel et al., 1991; Bucci et al., 1992). In yeast, two members of the Rab family, YPTl (Segev et al., 1988) and SEC4 (Salminen and Novick, 1987), are required for ER to Golgi and Golgi to plasma membrane transport, respectively. Based on a variety of studies, it has long been postulated that the Rab family of proteins defines the specificity of intracellular membrane trafficking (Bourne, 1988). However, recent studies in yeast in which domains between YPTl and SEC4 were exchanged suggest that these proteins are not exclusively responsible for membrane transport specificity (Brennwald and Novick, 1993; Dunn et al., 1993). Another candidate vesicle address marker is the synaptic vesicle-associated membrane protein (VAMP) (synaptobrevin;Trimbleetal., 1988; Elferinketal., 1989; Baumert et al., 1989). VAMP has recently been shown to be involved in the regulation of neurotransmitter release (Schravo et al., 1992). Several VAMP-related proteins have been identified outside the nervous system (Cain et al., 1992; Chin et al., 1993) including four proteins in yeast implicated in the regulation of multiple stages of the yeast secretory pathway(Dascher et al., 1991; Shim et al., 1991; J. Gerst, personal communication). Aconnection between the putative vesicle address marker VAMP and the target membrane receptor syntaxin is provided by the recent observation that both of these proteins can form a complex with two soluble factors, NSF and SNAP, required for transport vesicle consumption at multiple stages of the secretory and endocytic pathways (SolIner et al., 1993). These results raise the possibility that an interaction between a VAMP and a syntaxin provides the specificity of transport vesicle/target membrane docking. Such an interaction might then promote the assembly or recruitment of general soluble factors such as NSF and SNAP, ultimately leading to membrane fusion. Additional components, including the Rab proteins, could also contribute to the specificity either at docking or in the later fusion steps. Superimposedon thisconstitutivefusion machinery must be regulatory elements that, for example, confer cal-

A Family of Syntaxin-Related 071

Proteins

cium sensitivity to the fusion that underlies neurotransmitter release or cell cycle sensitivity to a variety of membrane trafficking events (Warren, 1989). The tools are now available to begin further dissecting the specificity of vesicle/ target membrane docking, to define the components of the fusion machinery that ultimately bring about bilayer fusion, and to address the mechanisms that underlie the physiological regulation of these events. Experimental

Procedures

Materials hZAPll cDNA libraries were obtained from Stratagene. Restriction enzymes and DNA modifying enzymes were from New England Biolabs and Eoehringer Mannheim. [a-32P]dCTP, [“S]dATPaS, and [“S]methionine were from DuPont-New England Nuclear. Tissue culture reagents were from the University of California, San Francisco, Cell Culture Facility, fetal bovine serum was from Gemini Bioproducts, and Permanox Z- and 8-chamber slides were from Nunc. Materials for SDS-polyacrylamide gel electrophoresis were from Bio-Rad. Glutathione-agarose, thrombin, and all other chemicals were purchased from Sigma. A genomic DNA clone of the Drosophila SED5 homolog was provided by I. Dawson (Yale University). Monoclonal antibody 12CA5 ascites fluid, directed against HA, was obtained from the Berkeley Antibody Company. Texas red-conjugated dextran and rhodamineconjugated concanavalin A were from Molecular Probes, and Citifluor antiquench reagent was from Citifluor Limited. Fluorescein-conjugated goat anti-mouse immunogobulin G (IgG), rhodamine-conjugated donkey anti-rabbit IgG secondary antibodies were from Chemicon, and fluorescein-conjugated goat anti-rabbit IgG secondary antibody was from TAGO. Monoclonal antibody HPC-1 directed against syntaxin (Inoue et al., 1992) was provided by C. Barnstable (Yale University), a rabbit polyclonal antiserum directed against DPH (Feng et al., 1992) was provided by R. Angeletti (Albert Einstein College of Medicine), and polyclonal antisera generated against B-COP and the mannose-6-phosphate receptor were provided by S. Pfeffer (Stanford University). Affinity-purified rabbit anti-syntaxin polyclonal antibody was prepared as previously described (Bennett et al., 1992). Library Screening and cDNA Sequencing Probes for screening cDNA libraries were prepared from syntaxin 1A and 18 either by polymerase chain reaction with primers flanking the respective coding sequences or by random hexamer-primed labeling of isolated cDNAfragments. Five cDNA libraries(from liver, brain stem/ spinal cord, macrophage, pituitary, and pancreas; 480,000 plaques each) were sceened with the syntaxin lA/l B-derived probes in 6 x SSC at 55%. From a number of potential positives, three distinct cDNA classes (in addition to syntaxin 1 Afl B) were identified (syntaxins 2-4). To identify a rat SEDdrelated cDNA, a probe was generated from a genomic DNA clone of the Drosophila SED5 homolog by polymerase chain reaction and used to screen a macrophage cDNA library (480,000 plaques) in 6x SSC at 50°C-55%. A single partial cDNA was isolated and subsequently used to isolate a full-length cDNA (syntaxin 5) from a rat brain cDNA library. Double-stranded DNA sequencing was performed using a Sequenase kit (U. S. Biochemicals) as described by the manufacturer. Utilizing T3, T7, and custom oligonucleotide primers, overlapping sequence was obtained from both strands of DNA over the entire coding region of each class of cDNA. In Vitro Transcription and In Vitro Translation Reagents for in vitro transcription and in vitro translation were obtained from Promega, and reactions were carried out as described by the manufacturer. In brief, a pBluescript II SK(-) plasmid (Stratagene) containing the full-length syntaxin 1A cDNA inserted into the EcoRl site was linearized with Notl and a syntaxin RNAtranscript synthesized with T7 RNA polymerase. The syntaxin transcript and a control a-factor transcript were in vitro translated in a rabbit reticulocyte lysate supplemented with [%]methionine either in the presence or absence of dog pancreatic microsomes. The translation product (1 ~1) was analyzed by SDS-polyacrylamide gel electrophoresis (12.5% polyacrylamide, 0.33% bisacrylamide). in some cases following treatment with 200 Kg/

ml proteinase K either in the presence or absence of 0.2% Triton X-100. The gels were processed for fluorography with Amplify (Amersham) prior to exposure to X-ray film. Northern Blots Northern blots (Clontech) contained poly(A)’ RNA isolated from eight different rat tissues (2 pgllane resolved on a denaturing formaldehyde agarose gel and transferred to nylon membrane). Probes were prepared either by hexamer-primed labeling (syntaxins IA, 2, 5, and b-actin) or by polymerase chain reaction (syntaxins lB,3, and 4). Blots were incubated with 5 x 108 to IO x 10ecpmlml of radiolabeled probe in 5 x SSPE at 42’C overnight, washed in 0.1 x SSC at 50°C-650C, and exposed to X-ray film.

Epitope Tagging and COS Cell Transfection A derivative of the Bluescript II KS(-) plasmid was constructed in which an ofigonucleotide encoding the HA epitope (MYPYDVPDYA) was directionally cloned into the Hindlll-Smal sites. Syntaxin cDNAs were inserted into this vector so that the HA epitope extended from the amino-terminal end of the syntaxin isoforms. The resulting constructs were then subconed into the COS cell expression vector pCMV (Andersson et al., 1989). COS cells were plated on two chamber slides (5 x lo4 cells per well) and grown for 1 day in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum, 100 U/ml penicillin, and 100 Kg/ml streptomycin. Transfection mix was prepared by mixing 50 ~1 of 2% DEAE-dextran with 10 Kg of recombinant pCMV plasmid DNA and then adding 1 ml of DMEM containing 100 PM chloroquine. The cultures were rinsed with DMEM and incubated with transfection mix at 37OC for 2-3 hr. The transfection mix was removed and replaced first with DMEM containing 10% dimethyl sulfoxide for 2.5 min and then with DMEM containing 10% fetal bovine serum, 100 U/ml penicillin, and 100 pg/ml streptomycin. After an overnight incubation, the medium was changed, and 48 hr later the cells were fixed and processed for immunofluorescence. lmmunofluorescence Microscopy Indirect immunofluorescence localization was performed on transiently transfected COS cells grown on Permanox P-chamber slides or nerve growth factor-induced PC1 2 cells grown on poly-lysine coated Permanox a-chamber slides. Cells were fixed with 4% paraformaldehyde in phosphate-buffered saline for 30 min, washed three times with phosphate-buffered saline containing 0.1 M glycine, and permeabilized with blocking buffer (phosphate-buffered saline containing 1% bovine serum albumin, 2% normal goat serum, and 0.4% saponin) for 5 min. The cells were incubated with primary antibody as indicated (anti-HA monoclonal antibody 12CA5 diluted 1:2000, anti-p-COP polyclonal antiserum diluted 1:500, or affinity-purified anti-syntaxin polyclonal antibody diluted 1:40) in blocking bufferfor l-2 hr, washed three times, and then incubated with the appropriate secondary antibody (fluorescein-conjugated goat anti-mouse IgG, rhodamine-conjugated donkey anti-rabbit IgG, or fluorescein-conjugated goat anti-rabbit IgG diluted 1 :lOO) in blocking buffer for l-2 hr. After washing three times, the slides were mounted with Citifluor and observed with a Zeiss Axiophot fluorescence microscope. For PC12 cell surface staining, the cells were incubated with primary antibody at 4OC for 1 hr in blocking buffer lacking saponin prior to fixation. COS cell plasma membrane staining was performed by incubating the cells in DMEM containing rhodamine-conjugatedconcanavalinA(l:l2S)for20 min at 15°C prior to fixation. The COS and PC1 2 cells were then washed and processed with primary or secondary antibody, respectively, as described above. PC12 Cell Microlnjection and Release Assay PC12 cell culture, microinjection, and detection of evoked DBH surface immunoreactivity were performed as previously described (Elferink et al., 1993). Samples for microinjection included a Fab fragment generated from an affinity-purified anti-syntaxin iA polyclonal antiserum (1 mglml; Bennett et al., 1992), a control F(ab’)2 fragment (2 mglml), Texas red-conjugated dextran (5 mglml, present in all samples), and bacterially expressed syntaxin IA fragments (2-3 mglml). Fusion proteins encoding cytoplasmic domains of syntaxin were prepared by insertion of syntaxin cDNA fragments in-frame with GST in the pGEXKG vector (Guan and Dixon, 1991). Following expression in Esche-

Cell 872

richra cok, the fusion protein was purified from the bacterial lysate by glutathione-agarose chromatography and thrombin cleavage as previously described (Bennett et al., 1992). A control preparation was prepared from bacteria transfected with the pGEX-KG vector with no Insert (GST only). All samples for microinjection were concentrated by ultrafiltration and dialyzed against microinjection buffer (Elferink et al , 1993). Acknowledgments The first three authors (M. K. B., J. E. G.-A., and L. A. E.) have contributed equally to this work. Correspondence should be addressed to R. H. S. We thank Colin Barnstable, Ruth Angeletti, and Suzanne Pfeffer for their kind gifts of antibodies, lain Dawson for providing the Drosophila SED5 DNA, and Sandy Bajjaliah for critical reading of the manuscript. J. E. G.-A. is on sabbatical leave from the University of Puerto Rico and is the recipient of a Minority Access to Research Careers-Nattonal Institute of General Medical Science faculty development fellowship. C. D. t-l. is supported by a National Science Foundation predoctoral fellowship. Financral support was provided by the National Institute of Mental Health. Recerved June 9, 1993; revrsed July 27, 1993. References Andersson. S., Davrs, D. L., Dahlback, H., Jornvall, H., and Russell, D. W (1989). Cloning, structure, and expression of the mitochondrial cytochrome P-450 sterol 26-hydroxylase, a bile acid biosynthetic enzyme. J. Brol. Chem. 264, 8222-8229. Baumert, M., Maycox, P. R., Navone, F., DeCamilli, P., and Jahn, Ft. (1989). Synaptobrevin: an Integral membraneproteinof 18,OOOdaltons present in small synaptic vesicles of rat brain. EMBO J. 8, 379-384. Bennett, M. K., and Scheller, Ft. H. (1993). The molecular machinery for secretion is conserved from yeast to neurons. Proc. Natl. Acad. Sci. USA 90, 2559-2563. Bennett, M. K., Calakos, N.. and Scheller, R. H. (1992). Syntaxin: a synaptic protein implicated in the docking of synaptic vesicles at presynaptic active zones. Science 257, 255-259. Bourne, H. (1988). Do GTPases direct membrane Cell 53. 669-671.

traffic in secretion?

Brennwald, P., and Novick, P. (1993). Interactions of three domains drstinguish the Ras-related GTP-binding proteins Yptl and Sec4. Nature 362, 560-563. Bucci, C., Parton, R. G., Mather, I. H., Stunnenberg, H., Simons, K., Hoflack, B., and Zerial, M. (1992). The small GTPase rab5 functions as a regulatory factor in the early endocytic pathway. Cell 70, 71% 728. Cain, C. C., Trimble, W. S., and Lienhard, G. E. (1992). Membersofthe VAMP family of synaptic vesicle proteins are components of glucose transporter-containing vesicles from rat adipocytes. J. Biol. Chem. 267, 11681-11684. Chavrrer, P., Parton, R. G., Hauri, H.-P., Simons, K., and Zerial, M. (1990). Localization of low molecular weight GTP binding proteins to exocyttc and endocytic compartments. Cell 62, 317-329. Chin, A., Burgess, R. W., Wong, B. R., Schwarz, T. L., and Scheller, R. H. (1993). Differential expression of transcripts from syb, a Drosophrla gene encoding VAMP that is abundant in non-neuronal cells. Gene, In press. Clift-O’Grady, L., Linstedt, A. D., Lowe, A. W., Grote. E., and Kelly, R. B. (1990). Biogenesisofsynapticvesicle-likestructures in pheochromocytoma PC-12 cells. J. Cell Biol. 770, 1693-1703.

associated membrane protein genes are differentially expressed in the rat central nervous system. J. Biol. Chem. 264, 11061-l 1064. Elferink, L. A., Anzai, K., and Scheller, R. H. (1992). rab15, a novel low molecular weight GTP-binding protein specifically expressed in rat brain. J. Biol. Chem. 267, 5768-5775. Elferink, L. A., Peterson, M. R., and Scheller, R. H. (1993). A role for synaptotagmin (~65) in regulated exocytosis. Cell 72, 153-l 59. Feng, Z., Angeletti, R., Levin, B. E., and Sabban, E. L. (1992). Glycosylation and membrane insertion of newly synthesized rat dopamine 6-hydroxylase in a cell-free system without signal cleavage. J. Biol. Chem. 267, 21808-21815. Field, J., Nikawa, J.-l., Broek, D., MacDonald, B., Rodgers, L., Wilson, I. A., Lerner, R. A., and Wigler, M. (1988). Purification of a RASresponsive adenylyl cyclase complex from Saccharomyces cerevisiae by use of an epitope tag addition. Mol. Cell. Biol. 8, 2159-2165. Fischer von Mallard, G., Mignery, G. A., Baumert, M.. Perin, M. S., Hanson, T. J., Burger, P. M., Jahn, R., and Stidhof, T. C:. (1990). rab3 is a small GTP-binding protein exclusively localized to synaptic vesicles. Proc. Natl. Acad. Sci. USA 87, 1988-1992. Gorvel, J.-P., Chavrier, P., Zerial, M.. and Gruenberg, J. (1991). rab5 controls an early endosome fusion event. Cell 64, 915-925. Guan, K. L., and Dixon, J. E. (1991). Eukaryotic proteins expressed in Escherichia co/i: an improved thrombin cleavage and purification procedure of fusion proteins with glutathione S-transferase. Anal. Biothem. 192, 262-267. Hardwick, K. G., and Pelham, H. A. B. (1992). SED5 encodes a 39-kD integral membrane protein required for vesicular transport between the ER and Golgi complex. J. Cell Biol. 779, 513-521. Hauri, H.-P., and Schweizer, A. (1992). The endoplasmic reticulumGolgi intermediate compartment. Curr. Opin. Cell Biol. 4, 600-608. Hirai, Y., Takebe, K., Takashina, M., Kobayashi, S., and Takeichi, M. (1992). Epimorphin: a mesenchymal protein essential for epithelial morphogenesis. Cell 69, 471-481. Hirai, Y., Nakagawa, S-i., and Takeichi, M. (1993). Reexamrnation of the properties of epimorphin and its possible roles. Cell 73, 426-427. Inoue, A., Obata, K., and Akagawa, K. (1992). Cloning and sequence analysis of cDNA for a neuronal cell membrane antigen, HPC-I. J. Biol. Chem. 267, 10613-10619. Jones, E. W. (1976). Proteinase mutants of Sacchafoomyces cerevisiae. Genetics 85, 23-33. Kelly, R. B. (1993). Storage and release of neurotransmttters. Neuron 70 (Suppl.), 43-53.

Cell 72/

Kyte, J., and Doolittle, R. F. (1982). A simple method for displaying the hydropathic character of a protein. J. Mol. Biol. 752, 105-132. Lippincott-Schwartz, J., Donaldson, E. G., Hauri, H.-P., Yuan, L. C., and bule-dependent retrograde transport presence of brefeldin A suggests an 821-836.

J. G., Schweizer, A., Berger, Klausner, Ft. D. (1990). Microtuof proteins into the ER in the ER recycling pathway Cell 60,

Morita, T., Mori, H., Sakimura, K.. Mishina, M., Sekine, Y., Tsugita, A., Odani, S., Horikawa, H. P. M., Saisu, H., and Abe, T. (1992). Synaptocanalin I, a protein associated with brain o-conotoxin-sensitive calcium channels. Biomed. Res. 73. 357-364. Orpins, A.. Duden, R., Kreis, T. E., Geuze, H. J.. and Slot, J. W. (1993). (&COP localizes mainly to the cis-Golgi side in exocrine pancreas. J. Cell Biol. 127, 49-59. Pelham, H. R. B. (1993). Is epimorphin Ceil 73, 425-426.

involved in vesrcular transport?

Salminen, A., and Novick. P. J. (1987). A fas-like protein is required for a post-Golgi event in yeast secretion. Cell 49, 527-538.

Dascher, C., Ossig, R.. Gallwitz, D., and Schmitt, H. D. (1991). Identification and structure of four yeast genes (SLY) that are able to suppress the functional loss of yPT1, a member of the RAS superfamily. Mol. Cell. Biol. 7 I, 872-885.

Schiavo, G., Benfenati, F., Poulain, B., Rossetto, O., Polverino de Laureto, P., DasGupta, B. R., and Montecucco, C. (1992). Tetanus and botulinurn-B neurotoxins block neurotransmitter release by proteotytic cleavage of synaptobrevin. Nature 359, 832-835.

Dunn, B.. Stearns, T., and Botstein, D. (1993). Specificity domains distinguish the Ras-related GTPases Yptl and Sec4. Nature 362,563565

Segev, N., Mulholland. J., and Botstein. D. (1988). The yeast GTPbinding YPTl protein and a mammalian counterpart are associated with the secretion machinery. Cell 52, 915-924.

Elferink, L. A., Trimble, W. S., and Scheller R. H. (1989). Two vesicle-

Shim, J., Newman, A. P., and Ferro-Novick,

S. (1991). TheSO.

gene

A Family of Syntaxin-Related 073

Proteins

encodes an essential 27-kD putative membrane protein that is required for vesicular transport from the ER to the Golgi complex in yeast, J. Cell Biol. 7 13, 55-64. Sdllner, T., Whiteheart. S. W., Brunner, M., Erdjument-Bromage, H., Geromanos, S., Tempst, P., and Rothman, J. E. (1993). SNAP receptors implicated in vesicle targeting and fusion. Nature 362, 318-324. Spring, J., Kato, M., and Bernfield, M. (1993). Epimorphin is related to a new class of neuronal and yeast vesicle targeting proteins Trends Biochem. Sci. 18, 124-125. Trimble, W. S., Cowan, D. M., and Scheller, R. H. (1988). VAMP-l: a synaptic vesicle-associated integral membrane protein. Proc. Natl. Acad. Sci. USA 85, 4538-4542. Warren, G. (1989). Mitosis and membranes. GenBank

Accession

Nature 342, 857-858.

Numbers

The accession numbers for the sequences reported in this paper are M95734 (syntaxin lA), M95735 (syntaxin lB), L20823 (synaxin 2), L20889 (syntaxin 2’) L20888 (syntaxin 2”) L20820 (syntaxin 3) L20821 (syntaxin 4) and L20822 (syntaxin 5).