Department of Physiology and Biophysics, The University of Iowa, Iowa City, Iowa 52242
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ABSTRACT |
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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
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INTRODUCTION |
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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 -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
-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.
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MATERIALS AND METHODS |
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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.
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RESULTS |
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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(102) was truncated at amino acid
102; EGFP-Syn5(
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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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(102), EGFP-Syn5(
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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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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DISCUSSION |
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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 -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.
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ACKNOWLEDGEMENTS |
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We thank Sarah Miller for care and maintenance of 3T3-L1 adipocytes.
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FOOTNOTES |
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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.
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