Transmembrane domain length determines intracellular membrane compartment localization of syntaxins 3, 4, and 5

Robert T. Watson and Jeffrey E. Pessin

Department of Physiology and Biophysics, The University of Iowa, Iowa City, Iowa 52242


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Insulin recruits glucose transporter 4 (GLUT-4) vesicles from intracellular stores to the plasma membrane in muscle and adipose tissue by specific interactions between the vesicle membrane-soluble N-ethylmaleimide-sensitive factor attachment protein target receptor (SNARE) protein VAMP-2 and the target membrane SNARE protein syntaxin 4. Although GLUT-4 vesicle trafficking has been intensely studied, few have focused on the mechanism by which the SNAREs themselves localize to specific membrane compartments. We therefore set out to identify the molecular determinants for localizing several syntaxin isoforms, including syntaxins 3, 4, and 5, to their respective intracellular compartments (plasma membrane for syntaxins 3 and 4; cis-Golgi for syntaxin 5). Analysis of a series of deletion and chimeric syntaxin constructs revealed that the 17-amino acid transmembrane domain of syntaxin 5 was sufficient to direct the cis-Golgi localization of several heterologous reporter constructs. In contrast, the longer 25-amino acid transmembrane domain of syntaxin 3 was sufficient to localize reporter constructs to the plasma membrane. Furthermore, truncation of the syntaxin 3 transmembrane domain to 17 amino acids resulted in a complete conversion to cis-Golgi compartmentalization that was indistinguishable from syntaxin 5. These data support a model wherein short transmembrane domains (<= 17 amino acids) direct the cis-Golgi localization of syntaxins, whereas long transmembrane domains (>= 23 amino acids) direct plasma membrane localization.

syntaxin; localization; membrane targeting


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

ACCORDING TO THE SOLUBLE N-ethylmaleimide-sensitive factor attachment protein target receptor (SNARE) hypothesis for membrane trafficking, vesicle membrane-soluble N-ethylmaleimide-sensitive factor attachment protein (SNAP) receptors (v-SNAREs) localized to vesicles interact with target membrane SNAP receptors (t-SNAREs) localized to target membranes (27-29). The formation of this v- and t-SNARE core complex results in a high-energy intermediate that is sufficient to catalyze bilayer fusion (37). Within the compartmentalized eukaryotic cell, a high degree of fusion specificity is required to maintain membrane compartment identity and to regulate the large volume of membrane lipid and protein traffic throughout the cell (5, 18, 23, 30). Although in vitro binding studies have observed that v- and t-SNAREs have the potential to interact promiscuously, under in vivo conditions specific pairing of SNARE partners most likely provides one layer of specificity for membrane fusion events (7, 31). Consistent with this notion, both v- and t-SNAREs comprise large families of proteins that localize to discrete membrane compartments within the cell. SNAREs may thus help to demarcate membrane compartments with the potential to participate in the fusion process.

Because their distribution within the cell may be critical for maintaining membrane compartment identity, several studies have investigated the mechanism by which SNAREs localize to specific membrane compartments. A specific signal within an alpha -helical domain of vesicle-associated membrane protein-2 (VAMP-2) was found to direct its localization to synaptic vesicles (14). In the case of syntaxin 5, an endoplasmic reticulum (ER) retrieval signal was found in the NH2-terminal extension of the 42-kDa (long) isoform (17). In addition, it was previously reported that a combination of cytosolic and transmembrane domain signals localizes the yeast and Drosophila homologues of syntaxin 5 (ySed5 and dSed5, respectively) to the cis-Golgi (2). More recently, two independent signals that cooperate to maintain syntaxin 6 in the trans-Golgi network (TGN) were identified, an alpha -helical retention motif that traps syntaxin 6 in the TGN and a retrieval signal that returns wayward syntaxin 6 molecules back to the TGN (36).

The above results are consistent with a model wherein SNARE proteins are actively maintained within specific membrane compartments. Indeed, the fidelity of bilayer fusion events may be ensured, at least in part, by sequestering the fusogenic SNARE proteins in spatially segregated compartments. Many proteins, including the insulin-responsive glucose transporter 4 (GLUT-4), navigate through multiple membrane compartments during their biogenesis, intracellular storage, exocytosis, and retrieval from the plasma membrane. Indeed, maintaining membrane compartment identity and fusion specificity is of critical importance because mislocalization or promiscuous fusion of GLUT-4 vesicles could compromise insulin responsivity. In this study, we have used differentiated 3T3-L1 adipocytes to investigate the localization mechanism of the abundant 35-kDa syntaxin 5 isoform (short form), which localizes to the cis-Golgi compartment, and syntaxins 3 and 4, which localize to the plasma membrane. Rather than a protein-targeting motif, we find that transmembrane domain length is sufficient to specify the cis-Golgi localization of syntaxin 5 and the plasma membrane localization of syntaxins 3 and 4.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Materials. Brefeldin A (Sigma) was prepared as a 5 mg/ml stock in methanol and used at a final concentration of 5 µg/ml. Cycloheximide (Sigma) was kept as a 10 mg/ml stock in ethanol and used at a final concentration of 10 µg/ml. Syntaxin 4 polyclonal antibody was obtained as described previously (22). Antibodies directed against syntaxin 5 and the Golgi-specific resident protein giantin were kind gifts from Dr. Tony Rowe (Metabolex, Hayward, CA) and Dr. Isabelle Moosbrugger (Institute of Immunology and Molecular Genetics, Karlsrue, Germany), respectively. The syntaxin 6 and immunoglobulin-binding protein/78-kDa glucose-regulated protein (BiP/grp78) monoclonal antibodies were purchased from Transduction Laboratories. Fluorescent secondary antibodies were purchased from Jackson Immunoresearch Laboratories.

Green fluorescent protein fusion constructs and syntaxin 4/syntaxin 5 chimera. To generate the NH2-terminal enhanced green fluorescent protein (EGFP) fusion constructs, the polymerase chain reaction (PCR) was used to introduce appropriate restriction enzyme sites on the 5' and 3' termini of cDNAs encoding syntaxins 3, 4, and 5. The PCR products were then cloned in-frame with the EGFP coding sequence of the pEGFP-C series vectors (Clontech). Syntaxin chimeras were generated using the PCR-based overlap extension method as described (16). Truncations of the syntaxin 3 and 5 cDNAs were generated by PCR using internal primers that hybridized to the specific region of interest. The PCR products were then cloned in-frame with EGFP.

Cell culture and transient transfection of 3T3-L1 adipocytes. Murine 3T3-L1 preadipocytes were purchased from the American Type Culture Collection repository. Cells were cultured in Dulbecco's modified Eagle's medium supplemented with 25 mM glucose and 10% calf serum at 37°C with 8% CO2. Cells were differentiated into adipocytes with 1 µg/ml insulin, 1 mM dexamethasone, and 0.5 mM 3-isobutyl-1-methylxanthine as previously described (16). Adipocytes were electroporated using the Gene Pulser II (Bio-Rad) with settings of 0.16 kV and 950 µF. Unless otherwise specified, 50 µg of DNA were used for electroporation. After electroporation, cells were plated on glass coverslips and allowed to recover for 20-48 h in complete medium before fixing.

Immunofluorescence and image analysis. Adipocytes expressing the EGFP fusion constructs were washed in phosphate-buffered saline (PBS) and fixed for 15 min in 4% paraformaldehyde containing 0.2% Triton X-100. Cells were washed in PBS and blocked in 5% donkey serum (Sigma) and 1% BSA (Sigma) for 1 h. Primary and secondary antibodies were used at 1:100 dilutions in blocking solution, and samples were mounted on glass slides with Vectashield (Vector Labs). Cells were imaged using confocal fluorescence microscopy. Images were then imported into Adobe Photoshop (Adobe Systems) for processing, and composite images were generated.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

EGFP-syntaxin reporter constructs. Syntaxin 5 is predicted to contain two helical domains, H1 and H2, that are thought to form coiled-coil interactions with other proteins. Similar to other syntaxin family members, syntaxin 5 also contains one transmembrane domain at the COOH terminus (Fig. 1). In contrast, based on the known structure of syntaxin 1, syntaxins 3 and 4 are each predicted to contain four helical domains, HA, HB, HC, and Hcore, as well as a COOH-terminal transmembrane domain (12, 33). As depicted in Fig. 1, we generated a panel of EGFP-tagged constructs, including full-length syntaxins 3, 4, and 5. In addition, we made a series of truncations of syntaxin 5, as well as a syntaxin 5/syntaxin 4 chimera. The construct designated EGFP-Syn5(Delta 102) was truncated at amino acid 102; EGFP-Syn5(Delta 220) was truncated at amino acid 220; and EGFP-Syn5(TMD) was truncated at amino acid 283. Chimera EGFP-Syn4(1-273)/Syn5(TMD) contains the NH2-terminal 273 amino acids of syntaxin 4 fused with the transmembrane domain of syntaxin 5. In addition, we also prepared two truncation mutants of syntaxin 3. EGFP-Syn3(TMD) was truncated at amino acid 253, and EGFP-Syn3(TMD17) contains the transmembrane domain of syntaxin 3 shortened down to 17 amino acids (residues 264-281). Finally, the Syn5(1-284)/Syn3(TMD) chimera contains amino acids 1-284 from syntaxin 5 fused to the transmembrane domain of syntaxin 3. 


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Fig. 1.   Schematic representation of enhanced green fluorescent protein (EGFP)-syntaxin reporter constructs used in this study. Shown are the syntaxin 5 sequence (open), syntaxin 4 (shaded), and syntaxin 3 (solid). EGFP was fused to the NH2 terminus of all constructs. The numbers in parentheses indicate the amino acid residues at which the proteins were truncated. For the Syn4(1-273)/Syn5(TMD) and Syn5(1-284)/Syn3(TMD) constructs, the numbers refer to the residues included in the chimera. The 25-amino acid transmembrane domain of syntaxin 3 was reduced to 17 residues in the Syn3(TMD17) construct. The H1, H2, HA, HB, HC, and Hcore domains are predicted alpha -helical motifs.

The transmembrane domain of syntaxin 5 is sufficient to confer cis-Golgi localization. As previously observed in fibroblasts (10), the endogenous syntaxin 5 protein (predominantly the 35-kDa short isoform) is concentrated in the perinuclear region of 3T3-L1 adipocytes, consistent with a Golgi localization pattern (Fig. 2a). Similarly, expression of EGFP-Syn5 also resulted in a perinuclear distribution similar to that of the endogenous syntaxin 5 protein (Fig. 2b). This localization pattern was essentially identical for the expressed EGFP-Syn5(Delta 102), EGFP-Syn5(Delta 220), and EGFP-Syn5(TMD) deletion constructs (Fig. 2, c-e). Because the transmembrane domain of syntaxin 5 also resulted in the same distribution as the endogenous syntaxin 5 protein, we next determined if this sequence was sufficient to confer perinuclear localization. We therefore expressed a chimeric protein that includes the cytosolic domain of syntaxin 4 fused to the transmembrane domain of syntaxin 5 (Fig. 2f). The Syn4(1-273)/Syn5(TMD) chimera was also distributed in the perinuclear region. This is in contrast to both endogenous syntaxin 4 protein and EGFP-Syn4, which were both predominantly localized to the plasma membrane (Fig. 2, g and h). Together, these data demonstrate that the syntaxin 5 transmembrane domain is sufficient to direct perinuclear localization of syntaxin 5. 


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Fig. 2.   Membrane compartment localization patterns of EGFP-syntaxin reporter constructs. Differentiated 3T3-L1 adipocytes were electroporated with 50 µg of the indicated EGFP-syntaxin reporter construct and visualized by confocal microscopy (see MATERIALS AND METHODS for details). Shown are representative fields from 3 or 4 independent determinations (original magnification, ×60).

The perinuclear region in adipocytes is composed of several distinct membrane compartments including the Golgi stacks (cis-, medial-, and trans-Golgi), the TGN, and recycling/sorting endosomes. To first verify that the syntaxin 5 transmembrane domain directed localization to the Golgi, we examined the colocalization of EGFP-Syn5 with the cis-Golgi marker protein, giantin (Fig. 3A). As typically observed, the endogenous giantin protein was localized in a compact juxtanuclear position (Fig. 3A, d-f). Similarly, adipocytes expressing EGFP-Syn5 also displayed a compact juxtanuclear distribution that completely overlapped with the endogenous giantin protein marker (Fig. 3A, b and c). Because expressed proteins can sometimes be trapped in the ER and undergo degradation, we colabeled cells with the ER marker BiP. As previously reported in fibroblasts (19), BiP showed a punctate distribution in 3T3-L1 adipocytes, with occasional peripheral clustering (Fig. 3A). In contrast to the extensive overlap with giantin, EGFP-Syn5 showed no overlap with BiP. The expressed Syn5(TMD) truncation had an identical distribution to the endogenous syntaxin 5 protein (Fig. 3B, a-c) and also did not overlap with BiP (Fig. 3B, d-f). Similarly, the Syn4(1-273)/Syn5(TMD) chimera overlapped very well with endogenous syntaxin 5 (Fig. 3C, a-c) but did not colocalize with BiP (Fig. 3C, d-f). These data are consistent with the transmembrane domain of syntaxin 5 imparting sequence information responsible for Golgi localization.


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Fig. 3.   The syntaxin 5 transmembrane domain is sufficient to confer Golgi localization. A: differentiated 3T3-L1 adipocytes were electroporated (50 µg DNA) with the cDNA encoding full-length EGFP-syntaxin 5 (a-f). The cells were then fixed and labeled with the giantin polyclonal antibody, followed by Texas red-conjugated secondary antibody (a) or the immunoglobulin-binding protein (BiP) monoclonal antibody (d), as described in MATERIALS AND METHODS. In the same field, the fluorescence of EGFP-Syn5 was also determined (b and e), and the merged images were obtained (c and f). B: differentiated 3T3-L1 adipocytes were electroporated (50 µg DNA) with the cDNAs encoding EGFP-Syn5(TMD) (a-f). The cells were then fixed and labeled with the syntaxin 5 polyclonal antibody (a) or the BiP monoclonal antibody (d) as described above. In the same field, the fluorescence of EGFP-Syn5 (TMD) was also determined (b and c). The merged images are shown in c and f. C: differentiated 3T3-L1 adipocytes were electroporated (50 µg DNA) with the cDNAs encoding EGFP-Syn4(1-273)/Syn5(TMD) (a-f). The cells were then fixed and labeled with the syntaxin 5 polyclonal antibody (a) or the BiP monoclonal antibody (d) as described above, and the merged images were obtained (c and f). In the same field, the fluorescence of EGFP-Syn4 (1-273)/Syn5 (TMD) was also determined (b and c). These are representative fields from 2 independent determinations (magnification, ×60).

Because immunofluorescence colocalization does not have sufficient resolution to distinguish between the Golgi stacks, TGN, and recycling/sorting endosomes, we took advantage of the fungal metabolite brefeldin A, which induces the collapse of the cis-Golgi membranes into the ER with only minimal morphological effects on the TGN or recycling/sorting endosome structures (3, 8). As previously observed, the endogenous syntaxin 5 protein displayed a perinuclear distribution characteristic of the Golgi (Fig. 4a). However, after treatment with brefeldin A, the syntaxin 5 protein was redistributed throughout the cell in small punctate structures reminiscent of the ER (Fig. 4b). Similarly, the EGFP-Syn5 fusion, the EGFP-Syn5(TMD) truncation, and the EGFP-Syn4(1-273)/Syn5(TMD) chimera all demonstrated a perinuclear localization that was dispersed in response to brefeldin A treatment (Fig. 4, c-h). As a control for the specificity of brefeldin A, syntaxin 6 has been established as a resident TGN protein (6, 36). Although syntaxin 6 shows a perinuclear labeling pattern very similar to that of syntaxin 5, treatment with brefeldin A had little effect on the localization of syntaxin 6 (Fig. 4, i and j). The more compacted appearance of syntaxin 6 is also consistent with the known property of brefeldin A to cause the TGN to coalesce into a more spherical mass near the centrioles. In any case, these data conclusively demonstrate that the syntaxin 5 transmembrane domain is responsible for defining cis-Golgi localization.


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Fig. 4.   The syntaxin 5 transmembrane domain confers specific localization to the cis-Golgi compartment. Differentiated 3T3-L1 adipocytes were labeled with a syntaxin 5 antibody (a and b), electroporated (50 µg) with the cDNA encoding the full-length EGFP-Syn5 (c and d), the EGFP-Syn5(TMD) (e and f), or the EGFP-Syn4(1-273)/Syn5(TMD) (g and h), or were labeled with a syntaxin 6 antibody (i and j). After an 18-h recovery period, the cells were then incubated either in the absence (a, c, e, g, and i) or in the presence (b, d, f, h, and j) of 5 µM brefeldin A for 1 h. The cells were fixed and subjected to confocal fluorescence microscopy as described in MATERIALS AND METHODS. These are representative fields of cells from 3 independent determinations (original magnification, ×60).

Transmembrane domain length determines cis-Golgi vs. plasma membrane compartmentalization. Inspection of the amino acid sequences for several of the mammalian syntaxin isoforms revealed that the plasma membrane-localized syntaxins (syntaxins 1, 2, 3, and 4) have relatively long transmembrane domains (23-25 residues), whereas the cis-Golgi-localized syntaxin 5 has a relatively short transmembrane domain (17 residues). In addition, no obvious amino acid sequence motif was discernible within the transmembrane domains of syntaxins localized to the cis-Golgi or cell surface (Fig. 5). To examine whether transmembrane domain length may be an important determinant in specifying plasma membrane vs. cis-Golgi compartmentalization, we next examined the properties of the plasma membrane-localized syntaxin 3 transmembrane domain. Expression of the EGFP-Syn3 fusion and Syn3(TMD) truncation proteins resulted in a predominant cell surface distribution (Fig. 6A, a and b). The trace amount of intracellular fluorescence observed with these two constructs most likely reflects either internalization of the reporter construct from the cell surface or exocytic transport of newly synthesized protein en route to the plasma membrane. In any case, expression of Syn3(TMD17) in which the syntaxin 3 transmembrane domain was truncated to 17 amino acids resulted in a complete loss of plasma membrane localization (Fig. 6A, c). Importantly, this construct was predominantly concentrated in the perinuclear region. Furthermore, the Syn3(TMD17) transmembrane domain truncation colocalized with the cis-Golgi marker giantin (Fig. 6B, a-c) but did not overlap with the ER marker BiP (Fig. 6B, d-f). It should be noted that we were unable to examine the effect of transmembrane domain length on syntaxin 4 localization, because truncations in this case resulted in misfolded proteins that did not become membrane inserted and instead remained cytosolic.


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Fig. 5.   Sequence alignment of the transmembrane domains of several syntaxin isoforms. Shown are the rat sequences for syntaxins 1-5 and 18 and the yeast sequences for Sed5, Sso1, and Sso2. The predicted transmembrane domains are underlined. Shown (right) is the predominant membrane compartment to which the isoforms localize. PM, plasma membrane; cG, cis-Golgi; ER, endoplasmic reticulum.



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Fig. 6.   Transmembrane domain length specifies Golgi vs. plasma membrane localization. A: differentiated 3T3-L1 adipocytes were electroporated (50 µg DNA) with the cDNAs encoding full-length EGFP-Syn3 (a), EGFP-Syn3(TMD) (b), or EGFP-Syn3(TMD17) (c). Cells were then fixed and processed for confocal microscopy as described in MATERIALS AND METHODS. B: differentiated 3T3-L1 adipocytes were electroporated (50 µg DNA) with the cDNA encoding EGFP-Syn3(TMD17) (a-f) and then fixed and labeled with the giantin polyclonal antibody (a) or the BiP monoclonal antibody (d). In the same field, the fluorescence of the EGFP-Syn3(TMD17) construct was also determined (b and e), and the merged images were obtained (c and f). C: differentiated 3T3-L1 adipocytes were electroporated (50 µg DNA) with the cDNA encoding EGFP-Syn5(1-284)/Syn3(TMD). At 18 h (a) and 48 h (b and c) following electroporation, cells were fixed and processed for immunofluorescence. The cells in c were treated with 10 µg/ml of cycloheximide (CHX) for 12 h, beginning 36 h after electroporation (original magnification, ×60).

In any case, to further verify that the transmembrane domain of syntaxin 3 was sufficient to direct plasma membrane localization, we generated a chimera wherein the transmembrane domain of syntaxin 5 was replaced with that of syntaxin 3, yielding the construct Syn5(1-284)/Syn3(TMD). This chimera also allowed us to investigate the possibility that sequence elements within the cytosolic domain of syntaxin 5 could participate along with its transmembrane domain in Golgi localization. Expression of the Syn5(1-284)/Syn3(TMD) reporter resulted in strong plasma membrane and perinuclear localization when cells were examined 18 h after transfection (Fig. 6C, a). However, incubation of cells expressing the Syn5(1-284)/Syn3(TMD) construct for 48 h after transfection, with or without the addition of cycloheximide, resulted in a significant loss of the reporter construct from the perinuclear region (Fig. 6C, b and c). These data indicate that the Syn5(1-284)/Syn3(TMD) chimera was efficiently chased to the plasma membrane under these conditions.

The localization of the Syn3(TMD17) construct to the cis-Golgi compartment was confirmed by brefeldin A treatment. Under these conditions, the reporter construct responded in a manner indistinguishable from syntaxin 5 (Fig. 7, a-d). The syntaxin 6 controls in this experiment are shown for comparison (Fig. 7, e and f). Together, these data demonstrate that the transmembrane domain of syntaxin 3 is sufficient to confer plasma membrane localization when it is 25 amino acids long. However, this domain also has the potential to confer cis-Golgi localization when reduced to 17 amino acids in length. Thus our data indicate that in the absence of specific overriding protein-protein interaction signals directing subcellular compartmentalization, transmembrane domain length alone is sufficient for the plasma membrane and cis-Golgi localization of syntaxins.


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Fig. 7.   The EGFP-Syn3(TMD17) construct localizes specifically to the cis-Golgi compartment. Differentiated 3T3-L1 adipocytes were labeled with a syntaxin 5 antibody (a and b), electroporated (50 µg) with the cDNA encoding the EGFP-Syn5(TMD17) construct (c and d), or labeled with a syntaxin 6 antibody (e and f). After an 18-h recovery period, the cells were then incubated either in the absence (a, c, and e) or in the presence of 5 µM brefeldin A for 1 h (b, d, and f). The cells were fixed and processed for confocal fluorescence microscopy as described in MATERIALS AND METHODS. These are representative fields of cells from 3 independent determinations (original magnification, ×60).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The endomembrane system of eukaryotic cells is organized into an array of functionally distinct sets of membrane-bound organelles. Protein transport among these compartments often occurs via vesicular shuttles, which bud from donor membranes, traffic to target membranes, and undergo a fusion process that results in bilayer mixing (21). The targeting and fusion of these vesicles requires a set of interacting proteins, collectively termed SNAREs, present on vesicle and target membranes. When vesicles reach their destination, cognate SNARE pairs interact in a parallel four-helix bundle that forms a bridge between vesicle and target membranes and is sufficient for fusion (37).

With respect to GLUT-4 vesicle trafficking, immunoelectron microscopy and subcellular fractionation studies have localized GLUT-4 to several compartments of the endomembrane system, including the TGN, clathrin-coated vesicles, and endosomes (26). However, the majority of GLUT-4 appears to reside in tubulovesicular elements in the cytoplasm that lie beneath the plasma membrane and may represent specialized GLUT-4 storage compartments. In addition, in the basal state, GLUT-4 cycles slowly between its intracellular storage compartment and the cell surface, where it undergoes endocytosis and is recycled back to its storage compartment. In the presence of insulin, the rate of GLUT-4 exocytosis is increased and the rate of GLUT-4 endocytosis is concomitantly decreased, resulting in a net redistribution of GLUT-4 to the cell surface. This complex trafficking itinerary requires multiple SNARE proteins, and to date, syntaxin 4, SNAP-23, VAMP-2, and VAMP-3 have been implicated in GLUT-4 vesicle trafficking (25, 26).

As originally conceived, the SNARE model proposed that membrane fusion specificity is achieved through specific interactions between v- and t-SNARE partners (27, 28, 32). However, recent evidence indicates that v- and t-SNAREs have the potential to interact promiscuously, as determined both by in vitro binding assays and in vivo overexpression studies (9, 11, 13, 35, 38). Although additional accessory proteins may help to maintain fusion specificity, t-SNAREs must be stable residents of specific membrane compartments, since mislocalized t-SNAREs would compromise membrane compartment identity through promiscuous vesicle fusions. Indeed, the localization of SNARE proteins to defined intracellular compartments could contribute substantially to membrane fusion specificity by spatially segregating the fusogenic SNARE molecules. Consistent with this notion, all known v- and t-SNARE proteins are localized to specific membrane compartments and are not randomly distributed (1, 4).

Previously we found that two independent signals in the cytosolic region of syntaxin 6, an alpha -helical domain and a YGRL motif, cooperate to localize this t-SNARE in the TGN (36). In this study, we demonstrate that the targeting determinants of the cis-Golgi and the plasma membrane syntaxins utilize the inherent property of transmembrane domain length rather than specific protein-interacting targeting motifs. The 17-amino acid syntaxin 5 transmembrane domain alone was sufficient to confer cis-Golgi localization when fused directly to EGFP. In addition, a heterologous syntaxin 4/syntaxin 5 chimera, in which the transmembrane domain of syntaxin 5 was fused to the cytosolic domain of syntaxin 4, also localized exclusively to cis-Golgi membranes. In contrast, the 25-amino acid transmembrane domain of syntaxin 3 was sufficient to confer plasma membrane localization. However, when the transmembrane domain of syntaxin 3 was truncated to 17 amino acids, this redirected syntaxin 3 to the cis-Golgi.

Several COOH-terminally anchored membrane proteins, including giantin, cytochrome b-5, and UBC6, have been shown to utilize transmembrane domain length for their membrane compartment localization (15, 20, 24, 39). Among SNARE family members, the yeast and Drosophila Sed5 proteins, which are syntaxin 5 homologues, were shown to localize to the cis-Golgi through a combination of cytosolic and transmembrane signals (2). It was found that a chimera containing the transmembrane domain of Sed5 and the cytosolic domain of syntaxin 2 (a plasma membrane syntaxin) was directed to the cis-Golgi. However, chimeras containing the cytosolic domain of Sed5 and the transmembrane domains from syntaxin 1B or syntaxin 2 were retained within the Golgi. This result suggests that the cytosolic domain of Sed5 may contribute to Golgi localization, although a specific targeting motif was not identified.

In contrast to the results previously obtained for Sed5, we found that a Syn5(1-284)/Syn3(TMD) chimera, which contains the cytosolic domain of syntaxin 5 fused to the transmembrane domain of syntaxin 3, showed strong plasma membrane localization. This chimera also showed some perinuclear localization when analyzed at 18 h posttransfection. However, the cytosolic domain of syntaxin 5 is known to interact with several SNARE proteins, including membrin and rbet1. Such interactions could have the effect of slowing the transit of the Syn5(1-284)/Syn3(TMD) construct through the Golgi. We therefore attempted to chase the reporter construct to the plasma membrane by allowing the cells to recover for 48 h after transfection or by preventing new protein synthesis with cycloheximide. Under both of these conditions, the majority of the Syn5(1-284)/Syn3(TMD) reporter was chased to the cell surface. Thus differences between our results and those reported previously for Sed5 may reflect a kinetic difference between these two systems. Nevertheless, our data clearly demonstrate that the Syn5(1-284)/Syn3(TMD) construct localized predominantly to the plasma membrane and only transiently occupied the Golgi en route to the cell surface.

Interestingly, mammalian cells express two syntaxin 5 isoforms, a 42-kDa long form and a 35-kDa short form. The 42-kDa isoform contains an ER retrieval signal (RKR) within the NH2-terminal extension that causes this isoform to partition between the ER and Golgi compartments in roughly equal amounts (17). Similarly, syntaxin 18 has a 17-amino acid transmembrane domain and localizes predominantly to the ER. In this case, syntaxin 18 also contains dilysine and diarginine sequences that may function as efficient ER retrieval signals (34). Thus these results suggest that transmembrane domain length plays a major role in membrane compartment localization, but protein-protein interaction motifs can evidently override the transmembrane localization mechanism in certain cases.

In summary, we demonstrate that the intracellular localization of the syntaxin 3, 4, and 5 type II membrane t-SNARE receptor proteins are specifically compartmentalized to the plasma membrane and cis-Golgi based on transmembrane domain length. Because SNARE proteins play key roles during vesicle fusion, their sequestration within specific subcellular compartments may contribute substantially to fusion specificity and compartment identity. Our results, therefore, relate directly to GLUT-4 and other vesicles that navigate through multiple membrane compartments during their regulated exocytic and endocytic processes.


    ACKNOWLEDGEMENTS

We thank Sarah Miller for care and maintenance of 3T3-L1 adipocytes.


    FOOTNOTES

This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-33823 and DK-25925.

Address for reprint requests and other correspondence: J. E. Pessin, Dept. of Physiology and Biophysics, Univ. of Iowa, Iowa City, IA 52242 (E-mail: Jeffrey-Pessin{at}uiowa.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 24 October 2000; accepted in final form 6 February 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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