Cell Biology Programme, The Hospital for Sick Children, Toronto, Ontario M5G 1X8; and Department of Biochemistry, University of Toronto, Toronto, Ontario, Canada M5S 1A8
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ABSTRACT |
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Twenty years ago it was shown that recruitment of glucose transporters from an internal membrane compartment to the plasma membrane led to increased glucose uptake into fat and muscle cells stimulated by insulin. The final step of this process is the fusion of glucose transporter 4 (GLUT-4)-containing vesicles with the plasma membrane. The identification of a neuronal soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex as a requirement for synaptic vesicle-plasma membrane fusion led to the search for homologous complexes outside the nervous system. Indeed, isoforms of the neuronal SNAREs were identified in muscle and fat cells and were shown to be required for GLUT-4 incorporation into the cell membrane. In addition, proteins that bind to nonneuronal SNAREs were cloned and proposed to regulate vesicle fusion. We have summarized the molecular mechanisms leading to membrane fusion in nonneuronal systems, focusing on the role of SNAREs and accessory proteins (Munc18c, synip, Rab4, and VAP-33) in incorporation of GLUT-4 into the plasma membrane. Potential modes of regulation of this process are discussed, including SNARE phosphorylation and interaction with the cytoskeleton.
vesicle traffic; soluble N-ethylmaleimide-sensitive factor attachment protein receptor; syntaxin 4; 23-kDa synaptosome-associated protein-like protein; vesicle-associated membrane protein 2
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DISCOVERY OF PROTEINS PARTICIPATING IN VESICLE FUSION |
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Vesicle-membrane fusion is a fundamental cellular
process that occurs at the final step of protein export to most
organelles and secretion of proteins and smaller molecules. Seminal
work from Rothman and colleagues (12, 20, 37, 125) in the
late 1980s identified a pair of soluble proteins that could bind to the
fusing membranes and were required for successful fusion of Golgi
vesicles with acceptor Golgi stacks. These proteins were termed
NSF (N-ethylmaleimide-sensitive factor) and SNAP (soluble NSF attachment protein) on the basis of the sensitivity of the former
to N-ethylmaleimide and the ability of both proteins to bind
to each other. Later, four membrane proteins from brain extracts were
found to act as receptors for NSF and SNAP and were termed SNAREs (for
SNAP receptors) (95). The proteins consisted of VAMP-2
(vesicle-associated membrane protein-2) (26), syntaxins A
and B (10), and SNAP-25 (25-kDa synaptosome-associated
proteins) (77). On the basis of their topological
localization in the presynaptic bouton, these proteins were classified
as vesicle (or v-) SNAREs (e.g., VAMP-2) and target (or t-) SNAREs
(e.g., syntaxin and SNAP-25) (Table 1).
VAMPs and syntaxins are characterized by a very short
extracellularly/luminally directed COOH terminus, a single
transmembrane domain, and a long cytoplasmic NH2-terminal region encompassing two coiled-coil domains (Fig.
1). In contrast, SNAP-25 does not have
transmembrane domains but presents two coiled-coil domains flanking a
cluster of cysteine residues that are highly susceptible to
palmitoylation (Fig. 1). The three proteins interact with one another
through their coiled-coil domains, and it is now thought that the
interaction of SNAP-25 with syntaxin is more relevant to its membrane
localization than is its palmitoylation (115, 116).
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It was originally proposed that the SNARE proteins would form a link
between vesicle and target membranes as a step preceding fusion
(95) and that fusion would be driven by the energy
released from ATP hydrolyzed by bound NSF (95).
Furthermore, given that individual SNARE isoforms were found to have
distinct tissular, cellular, and organellar specificity, it was
proposed that the SNAREs would dictate vesicle targeting specificity
(95). This hypothesis is supported by very recent work
showing that only certain SNARE isoforms are able to recover disrupted
norepinephrine release from cracked PC12 cells (89). It is
now clear that syntaxin and SNAP-25 also populate synaptic vesicles and
that VAMP is also found in target membranes (121). This
has led to the suggestion that cis-complexes of v- and
t-SNARE may occur within the same membrane, preventing the individual
components from engaging in trans-interactions with the
opposite membrane. The action of NSF and SNAP is to dissociate the
cis-complexes using the energy released by ATP hydrolysis
(Fig. 2) (7, 21). The final
fusion step depends on SNARE protein integrity but appears to be
independent of ATP hydrolysis, suggesting that NSF is not involved at
this stage (7, 21). A structure-function model of fusion
has been proposed whereby SNAREs in the docked conformation "zip
up" (Fig. 2) to form a tight, stable SNARE complex (40).
The complex involves a four-helix coiled-coil bundle now described at
atomic resolution (98). The free energy released by the
formation of this exceptionally stable complex is thought to be the
source of the energy used to fuse the two lipid bilayers
(40). The elucidation of the crystal structure of the
SNARE complex led to an alternative classification of SNAREs into Q- or
R-SNAREs, based on the presence of either a glutamine (Q) or arginine
(R) residue in the center of the SNARE complex (29).
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Because SNARE proteins were first identified in neuronal or neuroendocrine tissues, most of our information on these protein families stems from studies in neuronal systems. However, SNARE proteins are found in all tissue and cell types. In the last decade, more than 9 VAMP isoforms, 19 syntaxin isoforms, and 3 SNAP-25 isoforms have been described across the animal and even the plant kingdoms (51). The conservation of the basic elements of these proteins has given rise to the tenet that these proteins must fulfill a universal role in the mechanism of vesicle-membrane fusion (for a comprehensive review, see Ref. 51).
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GLUT-4 COMPARTMENTS AND THEIR SNARE ISOFORMS |
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An important biological phenomenon involving vesicle-membrane fusion is the incorporation of glucose transporters into the plasma membrane of muscle and fat cells. The glucose transporter of these tissues is GLUT-4, a 12-transmembrane domain protein that mediates vectoral transport of glucose in the direction of the glucose gradient (8). The hormone insulin strongly promotes GLUT-4 incorporation into the cell surface, and this translocation appears to fail in insulin resistance accompanying several forms of diabetes (60, 62, 130). Because of the physiological importance of insulin-dependent GLUT-4 translocation to the cell surface, attempts have been made to characterize the final GLUT-4 vesicle fusion step, drawing from lessons learned from neuronal synaptic transmission.
In unstimulated muscle and fat cells, the steady-state distribution of GLUT-4 favors intracellular compartments over the plasma membrane (25, 46, 52, 83). This steady state is the result of a slow mobilization of GLUT-4 to the cell surface and rapid removal from the plasma membrane (47, 53). Most studies suggest that the intracellular compartments populated by GLUT-4 include the early/sorting endosome, the recycling endosome, and a specialized vesicular compartment (41, 55, 56, 69). It is currently debated whether the latter does or does not recycle in the basal state. In rodent adipocytes, insulin promotes the externalization of the specialized vesicles and increases the recycling of GLUT-4 from the recycling endosome to the plasma membrane (41, 55, 56, 69). In muscle, insulin mobilizes a specialized vesicle pool, but there is no evidence that the recycling endosome is also mobilized (1, 2). Instead, emerging studies are consistent with the possibility that muscle contraction mobilizes GLUT-4 from the recycling endosome in this tissue (24, 63, 80). Thus GLUT-4-containing vesicles incorporate into the plasma membrane in at least three circumstances: in the basal (unstimulated) state, out of the recycling endosome; in response to insulin, out of the specialized vesicle; and in response to exercise in muscle and to insulin in fat cells, out of the recycling endosome. Other possibilities are not discounted, such as additional mobilization of the specialized vesicular pool in response to exercise. This diversity of fusion events begs the question of whether similar or different molecules participate in GLUT-4 vesicle fusion with the plasma membrane in each case. In the search for answers to this question, the SNARE isoforms expressed in muscle and fat cells had first to be defined. Of the VAMP family, only VAMP-2 and VAMP-3/cellubrevin have so far been detected in muscle and fat primary tissues and corresponding cells in culture (82, 105, 117). Contrary to neuronal and neuroendocrine cells, muscle and fat cells do not express syntaxin 1, but instead express syntaxin 4 (49, 102, 119). In addition, low levels of syntaxin 2 are also detected in 3T3-L1 adipocytes (119) and rat adipocytes (105), whereas small levels of syntaxin 3 are present in rat adipocytes (105). Another difference between neuronal/neuroendocrine cells and muscle and fat cells pertains to the expression of SNAP-25. This isoforms was not found in insulin-sensitive cells, which instead express SNAP-23 (3, 122, 127).
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THE PREFUSION STEPS |
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Synaptic vesicle fusion is the final step in a series of events that have been described as vesicle tethering (a reversible step) and vesicle docking (an irreversible step) (Fig. 2). Information on these steps has also emerged from yeast molecular genetic studies (16) and from in vitro endosome-endosome fusion studies (19, 71). The full complement of tethering proteins has not yet been identified, but the early endosome autoantigen 1 (EEA1) protein may act as the tethering protein engaged in binding endocytic vesicles to the early endosome (19, 70). EEA1 is found in insulin-sensitive cell types (78), but its participation in GLUT-4 vesicle traffic has not been tested.
Once vesicles are brought into close proximity with their target membranes by the tethering process, SNARE proteins on opposite membranes associate and acquire the configuration required for fusion (Fig. 2, "docked"). The SNARE proteins in docked vesicles are thought to be in a high-energy state (40) that can be maintained for long periods of time. Indeed, large numbers of synaptic vesicles can be observed docked at the presynaptic plasma membrane (51). In contrast, GLUT-4 vesicles have rarely, if at all, been found perched at the plasma membrane of unstimulated muscle or fat cells.
In neurons, nerve terminal depolarization leading to calcium influx is the penultimate trigger for neurotransmitter exocytosis. Currently, there is no evidence supporting a need for calcium ions in insulin-dependent glucose uptake mediated by GLUT-4 in 3T3-L1 adipocytes, L6 myotubes, or cardiac myocytes (42, 59, 61). In fact, GLUT-4 insertion into the plasma membrane can be observed in cells equilibrated with calcium-free buffers by means of streptolysin O-induced cell permeabilization (17) (Foster LJ and Klip A, unpublished observation).
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PROTEINS PARTICIPATING IN GLUT-4 VESICLE FUSION |
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VAMP-2
VAMP-2, the prototypical v-SNARE, is common to several systems in which vesicle traffic is regulated. These include neurotransmitter release in neural synapses (26), insulin-stimulated GLUT-4 translocation in fat and muscle cells (15, 117), and aquaporin-2 translocation in renal collecting ducts (54). VAMP-2 is expressed in muscle (82, 117) and fat cells (15, 118) and was originally detected in immunoisolated GLUT-4 compartments from rat fat cells (15). By subcellular fractionation of muscle and adipose cells, the protein is found to be distributed in similar proportions in the plasma membrane and intracellular membranes (15, 117, 118).VAMP-2 is susceptible to cleavage by various clostridium neurotoxins
(50). This susceptibility afforded a specific strategy to
probe the function of VAMP-2 (and a closely related isoform called
VAMP-3/cellubrevin) in GLUT-4 traffic. We and others have demonstrated
a requirement for VAMP-2 in insulin-stimulated GLUT-4 translocation
(17, 18, 31, 39, 65, 68, 75, 85, 99). Tetanus toxin and
botulinum toxins B and D introduced into rodent adipocytes by
electroporation, single-cell microinjection, chemical permeabilization
(using streptolysin O toxin), or natural, toxin-mediated uptake
(17, 18, 31, 39, 65, 99) reduced by more than one-half the
insulin-stimulated GLUT-4 incorporation into the cell surface (Table
2). In addition, introduction of antibodies raised against various regions of VAMP-2 as well as peptides
representing different segments of VAMP-2 also diminished the
insulin-dependent arrival of GLUT-4 at the plasma membrane of rodent
adipocytes (17, 65, 68, 75) (Table 2).
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Recent work on the function of VAMPs in GLUT-4 traffic has focused on resolving whether VAMP-2 or VAMP-3/cellubrevin is the primary v-SNARE involved in insulin-stimulated GLUT-4 translocation. It has been suggested that VAMP-2 is the v-SNARE important for translocation of GLUT-4 from the insulin-sensitive compartment, because the cytosolic domain of VAMP-2, but not VAMP-3/cellubrevin or VAMP-1, reduced insulin-stimulated GLUT-4 translocation by one-half when microinjected into 3T3-L1 adipocytes (68). In addition, transfection of tetanus toxin light chain into L6 muscle cells in culture resulted in 70% inhibition of insulin-dependent GLUT-4 arrival at the cell surface (85). Basal levels of cell surface GLUT-4 were minimally affected. Cotransfection of tetanus toxin-insensitive mutants of VAMP-2, but not VAMP-3, rescued the inhibition (85). These results indicate that VAMP-2, but not VAMP-3, is involved in insulin-stimulated GLUT-4 translocation and that neither protein participates in GLUT-4 sorting to the plasma membrane in the basal state.
Syntaxin 4
Syntaxin 4 is expressed in muscle and fat cells, where it is largely, but not exclusively, located at the plasma membrane (97, 105, 119). In fact, GLUT-4 vesicles contain syntaxin 4 that cannot be explained by contamination from plasma membranes (119). Unlike syntaxins 1 through 3, syntaxin 4 is not susceptible to cleavage by botulinum toxin C1 (90). For this reason, studies on the functional role of syntaxin 4 in GLUT-4 translocation have required the use of antibodies and peptides to perturb the function of syntaxin 4. Microinjection (65, 75, 101), chemical permeabilization (17, 119), and adenoviral overexpression (75) have been used to introduce antibodies directed against syntaxin 4 or soluble domains of the protein. In all cases, the perturbation of syntaxin 4 resulted in ~50% inhibition of insulin-stimulated glucose uptake (119) or GLUT-4 translocation (17, 65, 75, 101) (Table 2).SNAP-23
SNAP-23 shares both sequence and structural homology with SNAP-25 (86). A protein cloned from a cDNA library of 3T3-L1 adipocytes, originally named syndet (122), was found to be the murine form of SNAP-23 (96). By subcellular fractionation of muscle and fat cells, SNAP-23 is found almost exclusively in the plasma membrane-enriched fraction (122, 127). Neutralizing antibodies as well as peptides encoding the NH2 or COOH termini of SNAP-23 have been introduced into 3T3-L1 adipocytes by microinjection, chemical permeabilization, and adenoviral transfection. All these reagents reduced the insulin-dependent arrival of GLUT-4 at the plasma membrane, although they did not inhibit it completely (31, 34, 58, 87). The clostridium neurotoxins Bo/NT A and E have been useful to probe the function of SNAP-25, but they have been less effective in targeting SNAP-23. Bo/NT E has been shown to cleave SNAP-23 in some species, notably the canine isoform (64). In some reports, the toxin was able to cleave the murine SNAP-23, concomitantly reducing GLUT-4 translocation (31). In other studies, the toxin was ineffective toward SNAP-23 (18, 66). Because SNAP-23 is not a transmembrane protein and can bind to native syntaxin 4, it was also possible to introduce into cells full-length SNAP-23 to test its function. The microinjected full-length protein enhanced both insulin-stimulated GLUT-4 translocation and glucose uptake (34). These results suggest that the endogenous SNAP-23 may be available for SNARE complex formation in limiting amounts. A very recent report (43) has defined that 3T3-L1 adipocytes have approximately three times more SNAP-23 than syntaxin 4 (1.15 × 106 copies of SNAP-23 per cell compared with 3.74 × 105 copies of syntaxin 4). The extent of availability of each of these proteins for SNARE complex formation is still to be determined, given that these proteins have several cellular partners. Exogenous SNAP-23 may enhance the rate of fusion of GLUT-4 vesicles with the plasma membrane by enhancing the formation of productive complexes with syntaxin 4 and VAMP-2 (Table 2).NSF and SNAP
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DO SNARES SUFFICE TO CAUSE GLUT-4 VESICLE FUSION? |
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The three SNAREs expressed in muscle and fat cells appear to be essential for a significant fraction of the insulin-stimulated GLUT-4 arrival at the cell surface. However, in all the experiments discussed above, interfering with VAMP-2, syntaxin 4, or SNAP-23 only partly inhibited insulin-stimulated GLUT-4 translocation, and the basal levels of plasma membrane GLUT-4 were not altered even after long time periods in the presence of the perturbing agent. This latter observation is especially surprising given that GLUT-4 is known to cycle dynamically to and from the membrane in the absence of insulin. These observations suggest that either different SNARE isoforms or other proteins mediate these two fusion events. Only one other VAMP has been detected in the relevant membranes of muscle and fat cells, VAMP-3/cellubrevin. However, complete hydrolysis of this protein by botulinum or tetanus toxin, or interference by peptides emulating NH2-terminal sequences of VAMP-3/cellubrevin, failed to affect either basal or insulin-mediated GLUT-4 arrival at the cell surface (68, 75, 85), whereas the analogous domain of VAMP-2 effectively inhibited one-half of the insulin action (68). It is conceivable that muscle and fat cells express other, toxin-insensitive VAMPs, which could potentially mediate the fusion events that are not accounted for by VAMP-2. Similarly, it is conceivable that syntaxin 2 or 3 could mediate fusion events because they each bind to VAMP-2 (28, 81).
The studies listed above support the notion that VAMP-2, syntaxin 4, and SNAP-23 are required for the incorporation of GLUT-4-containing vesicles into the plasma membrane. These proteins may participate in the actual membrane fusion step, by analogy to the fusogen role assigned to their neuronal counterparts (51). Indeed, purified, bacterially expressed SNAP-25, syntaxin 1, and VAMP-2 reconstituted into synthetic proteoliposomes can mediate fusion of these liposomes (73, 124). Fusion of a single vesicle with its target membrane likely requires the formation of more than one SNARE complex, and there is a suggestion that a ring of SNARE complexes aligns around the fusion pore (124). Current experiments have not been able to determine the number of complexes required for fusion of one vesicle. This will likely require detailed microcalorimetry experiments measuring the free energy released during complex formation.
The importance of the formation of a neuronal SNARE complex for
synaptic vesicle fusion suggests that a high-affinity complex may also
form between VAMP-2, syntaxin 4, and SNAP-23 in the process of GLUT-4
vesicle fusion. This possibility has been addressed experimentally,
yielding somewhat surprising results (Table
3). Although there is general agreement
that a complex does form comprising SNAP-23, syntaxin 4, and VAMP-2,
the biochemical properties of such a complex appear to differ from
those of the VAMP-2/syntaxin 1/SNAP-25 complex. These differences
depend on the experimental design including, importantly, whether the
proteins used are recombinant forms produced in bacteria or endogenous
proteins isolated from mammalian cell systems. One of the hallmarks of
the neuronal SNARE complex involving SNAP-25, syntaxin 1, and VAMP-2 is
its ability to resist denaturation by ionic detergents such as sodium
dodecyl sulfate (SDS). Whereas an SDS-resistant complex containing
VAMP-2, SNAP-23, and syntaxin 4 in 0.5% SDS was detected using surface plasmon resonance (87), such a complex could not be
detected by SDS-PAGE or circular dichroism using samples in 2% SDS
(35, 128). A second distinguishing feature of the neuronal
SNARE complex is that SNAP-25 enhances the binding of VAMP-2 to
syntaxin 1. In contrast, in glutathione
S-transferase-pulldown experiments, we found no evidence for
cooperative binding between SNAP-23, VAMP-2, and syntaxin 4 (35); however, a cooperative effect of SNAP-23 on complex
formation was reported when all three proteins were overexpressed in
COS cells (58).
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HORMONAL REGULATION OF GLUT-4 VESICLE INCORPORATION INTO TARGET MEMBRANES |
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An important question is whether any of the steps involved in GLUT-4 vesicle incorporation into the plasma membrane is regulated by insulin-derived signals. The occupied insulin receptor undergoes autophosphorylation and then phosphorylates insulin-receptor substrate(s) (27, 69). This phosphorylation leads to activation of phosphatidylinositol 3'-kinase to produce phosphatidylinositol 3,4-bisphosphate and phosphatidylinositol 3,4,5-trisphosphate. These two lipids lead to the activation of protein kinase B/Akt and the atypical protein kinase C's (27, 32, 69), both of which appear to be required for GLUT-4 translocation (5, 6, 31, 44, 123). The steps downstream of these serine/threonine kinases leading to GLUT-4 translocation are as yet unidentified.
Recent studies have suggested that certain physiological conditions may halt GLUT-4 vesicles at the docking state and prevent fusion. Isoproterenol pretreatment reduces insulin-dependent glucose uptake in adipocytes and the number of GLUT-4 molecules detected at the surface of intact cells with the use of an impermeant photolabel (114). However, isolated plasma membranes did not reflect any diminution in GLUT-4 levels caused by isoproterenol (114). These results led to the suggestion that isoproterenol maintained GLUT-4 in an occluded state, inaccessible to the cell surface. Because the photolabel used has a preferential reactivity with active transporters, it was also possible that the transporters were inactive but present at the outer surface of the membrane. Therefore, it was important to detect transporter exposure at the cell surface by other means. With the use of cell-surface biotinylation, it was confirmed that isoproterenol pretreatment rendered the transporter inaccessible to extracellular labels yet bound to plasma membranes upon their isolation (30). These results suggest that isoproterenol pretreatment allows GLUT-4 vesicles to dock but prevents their fusion with the plasma membrane. Preliminary results from our laboratory suggest that isoproterenol may have similar effects in muscle cells. Treatment of L6 skeletal muscle cells with isoproterenol before insulin treatment reduced the insulin-stimulated glucose uptake. This was concomitant with a decrease in the insulin-stimulated GLUT-4 translocation measured by the externalization of an myc epitope inserted in the first extracellular loop of GLUT-4 and stably transfected into these cells (Hayashi M, Bilan P, and Klip A, unpublished observations). Experiments are currently underway to determine whether isoproterenol causes GLUT-4 vesicles to associate but not fuse with the plasma membrane in these cells as well.
It will be interesting to confirm, by using electron microscopy or in vitro reconstitution assays, that GLUT-4 vesicles can be arrested at the docked state. To date, the only reconstitution assay achieved detects GLUT-4 binding to the membrane but does not differentiate between docking and fusion (48). GLUT-4 vesicles from a 3T3-L1 adipocyte cell line expressing myc-tagged GLUT-4 were able to associate in vitro with isolated plasma membrane derived from wild-type 3T3-L1 cells. The association was dependent on calcium and could be prevented by including the recombinant soluble domain of syntaxin 4 in the binding assay. Notably, plasma membranes derived from insulin-stimulated cells were more effective than control membranes in binding intracellular GLUT-4 myc (48). Although this assay does not distinguish between docking and fusion, it may prove helpful in testing the individual participation of enzymes known to be activated by insulin on the interaction of donor and acceptor membranes.
Phosphorylation
A plausible mechanism whereby insulin- or isoproterenol-dependent signals could regulate the fusion machinery is through phosphorylation of its integral components. Indeed, considerable efforts have been dispensed toward defining which SNAREs are susceptible to phosphorylation and by which kinases (Table 4). We have reported that syntaxin 4 is susceptible to phosphorylation by the serine/threonine kinases protein kinase A, casein kinase II, and conventional protein kinase C in vitro (35). SNAP-23 is also phosphorylated by conventional protein kinase C's, but the phosphorylation is inefficient (35). In addition, the newly identified SNAP-23 kinase (SNAK) can phosphorylate SNAP-23 and syntaxin 4 (14).
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To further explore whether syntaxin 4, VAMP-2, or SNAP-23 is a suitable substrate of kinases in vivo, 3T3-L1 adipocytes were loaded with [o-32P]phosphate and treated with insulin, and each SNARE was selectively immunoprecipitated. Although each protein was found to be phosphorylated, the level was not altered significantly by insulin treatment. We further explored whether isoproterenol may alter SNARE phosphorylation, to provide a possible explanation for the inability of GLUT-4 vesicles to dock with the plasma membrane. However, isoproterenol did not significantly alter the phosphorylation levels of either SNAP-23 or syntaxin 4 (Foster LJ and Klip A, unpublished observations) (Table 4).
Ancillary Proteins
A second mechanism whereby SNARE function might be controlled is through binding of ancillary proteins that may either prevent or promote productive SNARE complex formation leading to membrane fusion. A cohort of proteins have been described to interact with SNAREs, as described below and listed in Tables 1 and 5.
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Munc18c.
The Munc18 proteins are mammalian homologs of the Sec1 protein in
Saccharomyces cerevisiae and the unc-18 protein in
Caenorhabditis elegans, both of which bind syntaxins from
their respective species. Munc18a was originally cloned from neuronal
tissues and has been given many different names, including n-Sec1 and
rbSec1. Munc18a cDNA was used as bait to clone similar genes from a
3T3-L1 cDNA library. One of the proteins cloned by this method was
Munc18c, which interacts specifically with syntaxins 2 and 4 but not
syntaxins 1 or 3 (100, 102). Munc18c inhibits the binding
of syntaxin 4 to VAMP-2 (101, 103) and SNAP-23
(3). Insulin causes the dissociation of a Munc18c/syntaxin
4 complex (103). A prediction of this observation is that
once insulin causes the dissociation, syntaxin 4 would be available to
bind SNAP-23 and VAMP-2, leading to fusion of the vesicles with the
target membrane. Indeed, full-length Munc18c introduced into 3T3-L1
adipocytes by adenoviral transfection inhibited insulin-stimulated
glucose uptake and GLUT-4 translocation by ~50% (100,
103). However, a peptide representing the domain of Munc18c that
binds to syntaxin 4, when microinjected into 3T3-L1 adipocytes,
inhibited fusion of green fluorescent protein-GLUT-4-containing vesicles with the plasma membrane. The peptide appeared to
allow GLUT-4 vesicles to dock with the plasma membrane without fusing with it. Given that this peptide displaces Munc18c-binding to syntaxin
4, these results may suggest that the displaced, endogenous Munc18c
catalyzes fusion (104) (Fig.
3).
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Synip. The recently cloned synip is a syntaxin 4-interacting protein, identified in a 3T3-L1 cDNA library by a yeast two-hybrid screen (72). Synip binding to syntaxin 4 prevents VAMP-2/syntaxin 4 binding but not SNAP-23/syntaxin 4 binding (72). As for Munc18c, the association of synip with syntaxin 4 is reduced in insulin-stimulated cells. Insulin-sensitivity is conferred by the NH2-terminal half of synip, whereas the COOH-terminal half modulates GLUT-4 translocation (72). Despite having unrelated primary sequences, synip and Munc18c regulate the availability of syntaxin 4 for fusion of GLUT-4 vesicles with the plasma membrane in response to insulin (Fig. 3). It will be interesting to determine whether the two proteins regulate different functional pools of syntaxin 4.
SNAK. SNAK is a protein kinase identified by its ability to bind syntaxin 4 in a yeast two-hybrid assay (14). However, SNAP-23 is a better substrate of SNAK than syntaxin 4 (14). SNAK phosphorylates SNAP-23 in vivo and in vitro, selectively phosphorylating only SNAP-23 that is not bound to syntaxin 4. SNAK phosphorylation of SNAP-23 enhances t-SNARE complex assembly, that is, binding of SNAP-23 and syntaxin 4 (14). It is unknown whether SNAK is present in insulin-sensitive tissues or whether SNAK is activated by insulin. Results of in vivo phosphorylation do not support any insulin-dependent phosphorylation of SNAP-23 (Table 4).
Hrs-2. The growth factor-induced phosphoprotein Hrs-2 can bind to SNAP-25 and SNAP-23 in vitro (110). In permeabilized PC12 cells, recombinant Hrs-2 inhibits norepinephrine release (9). Hrs-2 is expressed in muscle and fat cells, but its tyrosine phosphorylation state is not altered in response to insulin (Yaworsky K, Foster LJ, and Klip A, unpublished observations). To date, there is no evidence for its participation as a regulator of GLUT-4 traffic.
Pantophysin. A ubiquitous homolog of the synaptic vesicle protein synaptophysin, termed pantophysin, has recently been cloned from several sources (13, 38). This protein is found on GLUT-4-containing vesicles from 3T3-L1 cells and, similarly to synaptophysin, binds VAMP-2 (13). Interestingly, although pantophysin itself was not phosphorylated, a 77-kDa phosphoprotein associates with pantophysin upon treatment of cells with insulin (13). This result suggests a potential regulation of pantophysin by insulin. Preliminary results from our laboratory suggest that pantophysin availability is required for GLUT-4 vesicle fusion (Foster LJ, Cheatham B, and Klip A, unpublished observations).
VAP-33. A 33-kDa VAMP-2-associating protein (VAP-33) was isolated from an Aplysia californica cDNA library through a yeast two-hybrid approach (94). A human homolog was identified soon thereafter (126). VAP-33 is a single-transmembrane domain protein with the bulk of the molecule in the cytosol. Two isoforms of VAP-33 (VAP-33A and VAP-33B) bind VAMP-2 in vitro (74). We have recently shown (33) that VAP-33 is present on immunopurified VAMP-2 vesicles from L6 myotubes and 3T3-L1 adipocytes. Interestingly, overexpression of VAP-33A inhibited insulin-stimulated GLUT-4 translocation, and this effect was rescued by co-overexpression of VAMP-2. In addition, anti-VAP-33 antibodies microinjected into 3T3-L1 adipocytes also inhibited GLUT-4 translocation (33). We hypothesize that, as in the case of Munc18c, the levels of VAP-33A in the cell are critical and that shifting the balance of VAP-33A/B either above or below the critical point can have adverse effects on vesicle traffic.
Rab proteins. Rab GTPases represent a family of >35 proteins that relay information upon binding in their GTP-bound form to downstream effectors. Rabs become membrane-associated via geranylgeranylation or farnesylation, and this posttranslational modification is required for their GTPase function, which terminates their function on effectors. By genetic complementation, Rab proteins have been implicated in vesicular traffic, specifically in the recognition of vesicles by target membranes (for review, see Ref. 91). Deletion of the yeast Rab4 (Sec4p) can be rescued by overexpression of specific t-SNAREs, and the Rab Ypt1p is required for v-SNARE-t-SNARE complex formation. An emerging model suggests that Rab proteins direct vesicle traffic through the recruitment of docking factors from the cytosol. Thus Sec4 binds to the yeast exocyst that links vesicles to bud membranes, Rab5 binds to EEA1 that links endocytic vesicles to early endosomes, and vesicular VPs21 binds to Vac1 that links to the t-SNARE Ppe12 on target vesicles (4).
To date, only Rab4 has been implicated in GLUT-4 traffic by virtue of its presence on immunopurified GLUT-4 compartments (23, 111). Insulin stimulation causes Rab4 geranylgeranylation, GTP loading (92), and dissociation from GLUT-4-containing endomembranes (22, 36). Introduction of wild type or mutants of Rab4 or a peptide representing the hypervariable region of Rab4 resulted in inhibition of insulin-stimulated GLUT-4 translocation (22, 93, 120). Interestingly, a link between the Rab-mediated vesicle docking and the actin-based cytoskeleton has been established. Rabphilin, a Rab3 effector, interacts with the actin-bundling proteinThe Cytoskeleton
The actin cytoskeleton has been repeatedly implicated in exocytic events, both as a barrier separating the docked from stored synaptic vesicles (in essence, limiting the active zone) and as a facilitator of granule exocytosis (11, 112). Recent studies reveal that secretory granules acquire a coat of actin before exocytosis (113). Actin filaments are dynamic and, in addition to separating active zones and coating granules, they constitute stress fibers and cortical networks. The latter form in response to growth factor stimulation involving the Rho-family protein Rac (88), and in insulin-sensitive muscle cells, they present as large submembranous three-dimensional structures (59, 109). We have recently shown that formation of cortical actin structures is required for GLUT-4 exocytosis. Specifically, the rapidly forming subcortical actin mesh contained GLUT-4 vesicles and insulin signaling molecules (59). Preventing cortical actin structure formation through transient expression of a dominant negative Rac mutant abrogated externalization of GLUT-4 (59). In nontransfected muscle cells, GLUT-4 is inserted into the membrane at sites of membrane ruffles supported by cortical actin structures (106). Notably, SNAP-23 and syntaxin 4 appear to concentrate at sites of contact of the actin mesh with the plasma membrane (Khayat K, Foster LJ, and Klip A, unpublished observations). In adipocytes, a requirement for an organized cytoskeleton in GLUT-4 traffic has also been demonstrated (76). It will be interesting to determine whether GLUT-4 vesicles, once delivered by the cytoskeleton to the vicinity of the plasma membrane, require Rab4 as the tethering molecule leading to SNARE complex formation. ![]() |
CONCLUSION |
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GLUT-4 vesicle fusion bears similarities to and differences from the fusion of synaptic vesicles with their respective target membranes. VAMP-2, syntaxin 4, and SNAP-23, found in muscle and fat cells, form a SNARE complex that is similar but not identical to its neuronal counterpart, constituted by VAMP-2, syntaxin 1, and SNAP-25. Two mechanisms of insulin-dependent incorporation of GLUT-4 vesicles into the plasma membrane have been identified: one requiring VAMP-2, syntaxin 4, and SNAP-23, and one independent of these proteins. Although the fusion step is likely to be regulated by the hormone, to date there is no evidence for regulation through SNARE phosphorylation. However, it is conceivable that subtle regulation may still occur by this means. In contrast, there is emerging evidence that ancillary proteins such as Munc18c, synip, VAP-33, and pantophysin may regulate the availability of VAMP-2 or syntaxin 4 for productive GLUT-4 fusion. Cytoskeletal tethering of vesicles and SNAP-23 may provide entropic energy to the process of GLUT-4 vesicle fusion. Future studies should reveal whether the fine tuning of GLUT-4 vesicle fusion is altered in insulin-resistant states leading to or accompanying diabetes. In this regard, two recent studies (67, 84) report increases in muscle SNARE protein levels in two animal models of insulin resistance. Both reports suggest that these increases might be adaptive changes attempting to overcome the defects in GLUT-4 traffic that underlie insulin resistance.
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ACKNOWLEDGEMENTS |
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We acknowledge the Juvenile Diabetes Foundation and the Medical Research Council of Canada for funding.
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FOOTNOTES |
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Address for reprint requests and other correspondence: A. Klip, Cell Biology Programme, The Hospital for Sick Children, Toronto, Ontario, Canada M5G 1X8 (E-mail: amira{at}sickkids.on.ca).
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REFERENCES |
---|
1.
Aledo, JC,
Darakhshan F,
and
Hundal HS.
Rab4, but not the transferrin receptor, is colocalized with GLUT4 in an insulin-sensitive intracellular compartment in rat skeletal muscle.
Biochem Biophys Res Commun
215:
321-328,
1995[ISI][Medline].
2.
Aledo, JC,
Lavoie L,
Volchuk A,
Keller SR,
Klip A,
and
Hundal HS.
Identification and characterization of two distinct intracellular GLUT4 pools in rat skeletal muscle: evidence for an endosomal and an insulin-sensitive GLUT4 compartment.
Biochem J
325:
727-732,
1997[ISI][Medline].
3.
Araki, S,
Tamori Y,
Kawanishi M,
Shinoda H,
Masugi J,
Mori H,
Niki T,
Okazawa H,
Kubota T,
and
Kasuga M.
Inhibition of the binding of SNAP-23 to syntaxin 4 by Munc18c.
Biochem Biophys Res Commun
234:
257-262,
1997[ISI][Medline].
4.
Armstrong, J.
How do Rab proteins function in membrane traffic.
Int J Biochem Cell Biol
32:
303-307,
2000[ISI][Medline].
5.
Bandyopadhyay, G,
Standaert ML,
Kikkawa U,
Ono Y,
Moscat J,
and
Farese RV.
Effects of transiently expressed atypical (zeta, lambda), conventional (,
) and novel (
,
) protein kinase C isoforms on insulin-stimulated translocation of epitope-tagged GLUT4 glucose transporters in rat adipocytes: specific interchangeable effects of protein kinases C-zeta and C-lambda.
Biochem J
337:
461-470,
1999[ISI][Medline].
6.
Bandyopadhyay, G,
Standaert ML,
Sajan MP,
Karnitz LM,
Cong L,
Quon MJ,
and
Farese RV.
Dependence of insulin-stimulated glucose transporter 4 translocation on 3-phosphoinositide-dependent protein kinase-1 and its target threonine-410 in the activation loop of protein kinase C-zeta.
Mol Endocrinol
13:
1766-1772,
1999
7.
Banerjee, A,
Barry VA,
DasGupta BR,
and
Martin TFJ
N-ethylmaleimide-sensitive factor acts at a prefusion ATP-dependent step in Ca2+-activated exocytosis.
J Biol Chem
271:
20223-20226,
1996
8.
Barrett, MP,
Walmsley AR,
and
Gould GW.
Structure and function of facilitative sugar transporters.
Curr Opin Cell Biol
11:
496-502,
1999[ISI][Medline].
9.
Bean, AJ,
Seifert R,
Chen YA,
Sacks R,
and
Scheller RH.
Hrs-2 is an ATPase implicated in calcium-regulated secretion.
Nature
385:
826-829,
1997[ISI][Medline].
10.
Bennett, MK,
Calakos N,
and
Scheller RH.
Syntaxin: a synaptic protein implicated in docking of synaptic vesicles at presynaptic active zones.
Science
257:
255-259,
1992[ISI][Medline].
11.
Bernstein, BW,
DeWit M,
and
Bamburg JR.
Actin disassembles reversibly during electrically induced recycling of synaptic vesicles in cultured neurons.
Mol Brain Res
53:
236-250,
1998[ISI][Medline].
12.
Block, MR,
Glick BS,
Wilcox CA,
Wieland FT,
and
Rothman JE.
Purification of an N-ethylmaleimide-sensitive protein catalyzing vesicular transport.
Proc Natl Acad Sci USA
85:
7852-7856,
1988[Abstract].
13.
Brooks, CC,
Scherer PE,
Cleveland K,
Whittemore JL,
Lodish HF,
and
Cheatham B.
Pantophysin is a phosphoprotein component of adipocyte transport vesicles and associates with GLUT4-containing vesicles.
J Biol Chem
275:
2029-2036,
2000
14.
Cabaniols, JP,
Ravichandran V,
and
Roche PA.
Phosphorylation of SNAP-23 by the novel kinase SNAK regulates t-SNARE complex assembly.
Mol Biol Cell
10:
4033-4041,
1999
15.
Cain, CC,
Trimble WS,
and
Lienhard GE.
Members of the VAMP family of synaptic vesicle proteins are components of glucose transporter-containing vesicles from rat adipocytes.
J Biol Chem
267:
11681-11684,
1992
16.
Cao, X,
Ballew N,
and
Barlowe C.
Initial docking of ER-derived vesicles requires Uso1p and Ypt1p but is independent of SNARE proteins.
EMBO J
17:
2156-2165,
1998
17.
Cheatham, B,
Volchuk A,
Kahn CR,
Wang L,
Rhodes CJ,
and
Klip A.
Insulin-stimulated translocation of GLUT4 glucose transporters requires SNARE-complex proteins.
Proc Natl Acad Sci USA
93:
15169-15173,
1996
18.
Chen, FS,
Foran P,
Shone CC,
Foster KA,
Melling J,
and
Dolly JO.
Botulinum neurotoxin B inhibits insulin-stimulated glucose uptake into 3T3-L1 adipocytes and cleaves cellubrevin unlike type A toxin which failed to proteolyze the SNAP-23 present.
Biochemistry
36:
5719-5728,
1997[ISI][Medline].
19.
Christoforidis, S,
McBride HM,
Burgoyne RD,
and
Zerial M.
The Rab5 effector EEA1 is a core component of endosome docking.
Nature
397:
621-625,
1999[ISI][Medline].
20.
Clary, DO,
Griff IC,
and
Rothman JE.
SNAPs, a family of NSF attachment proteins involved in intracellular membrane fusion in animals and yeast.
Cell
61:
709-721,
1990[ISI][Medline].
21.
Colombo, MI,
Taddese M,
Whiteheart SW,
and
Stahl PD.
A possible predocking attachment site for N-ethylmaleimide-sensitive fusion protein. Insights from in vitro endosome fusion.
J Biol Chem
271:
18810-18816,
1996
22.
Cormont, M,
Bortoluzzi MN,
Gautier N,
Mari M,
van Obberghen E,
and
Le Marchand-Brustel Y.
Potential role of Rab4 in the regulation of subcellular localization of Glut4 in adipocytes.
Mol Cell Biol
16:
6879-6886,
1996[Abstract].
23.
Cormont, M,
Tanti JF,
Zahraoui A,
van Obberghen E,
Tavitian A,
and
Le Marchand-Brustel Y.
Insulin and okadaic acid induce rab4 redistribution in adipocytes.
J Biol Chem
268:
19491-19497,
1993
24.
Cushman, SW,
Goodyear LJ,
Pilch PF,
Ralston E,
Galbo H,
Ploug T,
Kristiansen S,
and
Klip A.
Molecular mechanisms involved in GLUT4 translocation in muscle during insulin and contraction stimulation.
Adv Exp Med Biol
441:
63-71,
1998[ISI][Medline].
25.
Douen, AG,
Ramlal T,
Rastogi S,
Bilan PJ,
Cartee GD,
Vranic M,
Holloszy JO,
and
Klip A.
Exercise induces recruitment of the "insulin-responsive glucose transporter". Evidence for distinct intracellular insulin- and exercise-recruitable transporter pools in skeletal muscle.
J Biol Chem
265:
13427-13430,
1990
26.
Elferink, LA,
Trimble WS,
and
Scheller RH.
Two vesicle-associated membrane protein genes are differentially expressed in the rat central nervous system.
J Biol Chem
264:
11061-11064,
1989
27.
Elmendorf, JS,
and
Pessin JE.
Insulin signaling regulating the trafficking and plasma membrane fusion of GLUT4-containing intracellular vesicles.
Exp Cell Res
253:
55-62,
1999[ISI][Medline].
28.
Fasshauer, D,
Antonin W,
Margittai M,
Pabst S,
and
Jahn R.
Mixed and non-cognate SNARE complexes. Characterization of assembly and biophysical properties.
J Biol Chem
274:
15440-15446,
1999
29.
Fasshauer, D,
Sutton RB,
Brunger AT,
and
Jahn R.
Conserved structural features of the synaptic fusion complex: SNARE proteins reclassified as Q- and R-SNAREs.
Proc Natl Acad Sci USA
95:
15781-15786,
1998
30.
Ferrara, CM,
and
Cushman SW.
GLUT4 trafficking in insulin-stimulated rat adipose cells: evidence that heterotrimeric GTP-binding proteins regulate the fusion of docked GLUT4-containing vesicles.
Biochem J
343:
571-577,
1999[ISI][Medline].
31.
Foran, PG,
Fletcher LM,
Oatey PB,
Mohammed N,
Dolly JO,
and
Tavare JM.
Protein kinase B stimulates the translocation of GLUT4 but not GLUT1 or transferrin receptors in 3T3-L1 adipocytes by a pathway involving SNAP-23, synaptobrevin-2, and/or cellubrevin.
J Biol Chem
274:
28087-28095,
1999
32.
Foster, LJ,
Khayat ZA,
and
Klip A.
Insulin-dependent intracellular traffic of glucose transporters.
In: The Diabetes Annual, edited by Marshall SM,
Home PD,
and Rizza RA.. Amsterdam: Elsevier Science, 1999, p. 111-140.
33.
Foster, LJ,
Weir ML,
Liu Z,
Trimble WS,
and
Klip A.
A regulatory role for VAP-33 in insulin-stimulated GLUT4 traffic.
Traffic
1:
512-521,
2000[ISI][Medline].
34.
Foster, LJ,
Yaworsky K,
Trimble WS,
and
Klip A.
SNAP23 promotes insulin-dependent glucose uptake in 3T3-L1 adipocytes: possible interaction with cytoskeleton.
Am J Physiol Cell Physiol
276:
C1108-C1114,
1999
35.
Foster, LJ,
Yeung B,
Mohtashami M,
Ross K,
Trimble WS,
and
Klip A.
Binary interactions of the SNARE proteins syntaxin-4, SNAP23, and VAMP-2 and their regulation by phosphorylation.
Biochemistry
37:
11089-11096,
1998[ISI][Medline].
36.
Goalstone, ML,
Leitner JW,
Golovchenko I,
Stjernholm MR,
Cormont M,
Le Marchand-Brustel Y,
and
Draznin B.
Insulin promotes phosphorylation and activation of geranylgeranyltransferase II. Studies with geranylgeranylation of rab-3 and rab-4.
J Biol Chem
274:
2880-2884,
1999
37.
Griff, IC,
Schekman R,
Rothman JE,
and
Kaiser CA.
The yeast SEC17 gene product is functionally equivalent to mammalian alpha-SNAP protein.
J Biol Chem
267:
12106-12115,
1992
38.
Haass, NK,
Kartenbeck MA,
and
Leube RE.
Pantophysin is a ubiquitously expressed synaptophysin homologue and defines constitutive transport vesicles.
J Cell Biol
134:
731-746,
1996[Abstract].
39.
Hajduch, E,
Aledo JC,
Watts C,
and
Hundal HS.
Proteolytic cleavage of cellubrevin and vesicle-associated membrane protein (VAMP) by tetanus toxin does not impair insulin-stimulated glucose transport or GLUT4 translocation in rat adipocytes.
Biochem J
321:
233-238,
1997[ISI][Medline].
40.
Hanson, PI,
Heuser JE,
and
Jahn R.
Neurotransmitter release - four years of SNARE complexes.
Curr Opin Neurobiol
7:
310-315,
1997[ISI][Medline].
41.
Hashiramoto, M,
and
James DE.
Characterization of insulin-responsive GLUT4 storage vesicles isolated from 3T3-L1 adipocytes.
Mol Cell Biol
20:
416-427,
2000
42.
Haworth, RA,
Hunter DR,
and
Berkoff HA.
Insulin stimulates deoxyglucose transport in adult rat heart cells in the absence of Ca2+.
FEBS Lett
141:
37-40,
1982[ISI][Medline].
43.
Hickson, GR,
Chamberlain LH,
Maier VH,
and
Gould GW.
Quantification of SNARE protein levels in 3T3-L1 adipocytes: implications for insulin-stimulated glucose transport.
Biochem Biophys Res Commun
270:
841-845,
2000[ISI][Medline].
44.
Hill, MM,
Clark SF,
Tucker DF,
Birnbaum MJ,
James DE,
and
Macaulay SL.
A role for protein kinase Bbeta/Akt2 in insulin-stimulated GLUT4 translocation in adipocytes.
Mol Cell Biol
19:
7771-7781,
1999
45.
Hinck, CS,
St-Denis JF,
Al-Hasani H,
Zarnowski MJ,
Whiteheart SW,
and
Cushman SW.
Expression of an ATPase-deficient mutant of N-ethylmaleimide sensitive fusion protein (NSF) in rat adipose cells impairs insulin-stimulated GLUT4 translocation.
Diabetes
47:
A241,
1998[ISI].
46.
Hirshman, MF,
Goodyear LJ,
Wardzala LJ,
Horton ED,
and
Horton ES.
Identification of an intracellular pool of glucose transporters from basal and insulin-stimulated rat skeletal muscle.
J Biol Chem
265:
987-991,
1990
47.
Holman, GD,
and
Cushman SW.
Subcellular localization and trafficking of the GLUT4 glucose transporter isoform in insulin-responsive cells.
Bioessays
16:
753-759,
1994[ISI][Medline].
48.
Inoue, G,
Cheatham B,
and
Kahn CR.
Development of an in vitro reconstitution assay for glucose transporter 4 translocation.
Proc Natl Acad Sci USA
96:
14919-14924,
1999
49.
Jagadish, MN,
Fernandez CS,
Hewish DR,
Macaulay SL,
Gough KH,
Grusovin J,
Verkuylen A,
Cosgrove L,
Alafaci A,
Frenkel MJ,
and
Ward CW.
Insulin-responsive tissues contain the core complex protein SNAP-25 (synaptosomal-associated protein 25) A and B isoforms in addition to syntaxin 4 and synaptobrevins 1 and 2.
Biochem J
317:
945-954,
1996[ISI][Medline].
50.
Jahn, R,
Hanson PI,
Otto H,
and
Ahnert-Hilger G.
Botulinum and tetanus neurotoxins: emerging tools for the study of membrane fusion.
Cold Spring Harb Symp Quant Biol
60:
329-335,
1995[ISI][Medline].
51.
Jahn, R,
and
Südhof TC.
Membrane fusion and exocytosis.
Annu Rev Biochem
68:
863-911,
1999[ISI][Medline].
52.
James, DE,
Brown R,
Navarro J,
and
Pilch PF.
Insulin-regulatable tissues express a unique insulin-sensitive glucose transport protein.
Nature
333:
183-185,
1988[ISI][Medline].
53.
Jhun, BH,
Rampal AL,
Liu H,
Lachaal M,
and
Jung CY.
Effects of insulin on steady state kinetics of GLUT4 subcellular distribution in rat adipocytes. Evidence of constitutive GLUT4 recycling.
J Biol Chem
267:
17710-17715,
1992
54.
Jo, I,
Harris HW,
Amendt-Raduege AM,
Majewski RR,
and
Hammond TG.
Rat kidney papilla contains abundant synaptobrevin protein that participates in the fusion of antidiuretic hormone-regulated water channel-containing endosomes in vitro.
Proc Natl Acad Sci USA
92:
1876-1880,
1995[Abstract].
55.
Kandror, KV.
Insulin regulation of protein traffic in rat adipose cells.
J Biol Chem
274:
25210-25217,
1999
56.
Kandror, KV,
and
Pilch PF.
Multiple endosomal recycling pathways in rat adipose cells.
Biochem J
331:
829-835,
1998[ISI][Medline].
57.
Kato, M,
Sasaki T,
Ohya T,
Nakanishi H,
Nishioka H,
Imamura M,
and
Takai Y.
Physical and functional interaction of rabphilin-3A with alpha-actinin.
J Biol Chem
271:
31775-31778,
1996
58.
Kawanishi, M,
Tamori Y,
Okazawa H,
Araki S,
Shinoda H,
and
Kasuga M.
Role of SNAP23 in insulin-induced translocation of GLUT4 in 3T3-L1 adipocytes. Mediation of complex formation between syntaxin4 and vamp2.
J Biol Chem
275:
8240-8247,
2000
59.
Khayat, ZA,
Tong P,
Yaworsky K,
Bloch RJ,
and
Klip A.
Insulin-induced actin filament remodeling colocalizes actin with phosphatidylinositol 3-kinase and GLUT4 in L6 myotubes.
J Cell Sci
113:
279-290,
2000
60.
King, PA,
Horton ED,
Hirshman MF,
and
Horton ES.
Insulin resistance in obese Zucker rat (fa/fa) skeletal muscle is associated with a failure of glucose transporter translocation.
J Clin Invest
90:
1568-1575,
1992[ISI][Medline].
61.
Klip, A,
and
Ramlal T.
Cytoplasmic Ca2+ during differentiation of 3T3-L1 adipocytes. Effect of insulin and relation to glucose transport.
J Biol Chem
262:
9141-9146,
1987
62.
Klip, A,
Ramlal T,
Bilan PJ,
Cartee GD,
Gulve EA,
and
Holloszy JO.
Recruitment of GLUT4 glucose transporters by insulin in diabetic rat skeletal muscle.
Biochem Biophys Res Commun
172:
728-736,
1990[ISI][Medline].
63.
Lemieux, K,
Han XX,
Dombrowski L,
Bonen A,
and
Marette A.
The transferrin receptor defines two distinct contraction-responsive GLUT4 vesicle populations in skeletal muscle.
Diabetes
49:
183-189,
2000[Abstract].
64.
Leung, SM,
Chen D,
DasGupta BR,
Whiteheart SW,
and
Apodaca G.
SNAP-23 requirement for transferrin recycling in streptolysin-O-permeabilized Madin-Darby canine kidney cells.
J Biol Chem
273:
17732-17741,
1998
65.
Macaulay, SL,
Hewish DR,
Gough KH,
Stoichevska V,
Macpherson SF,
Jagadish M,
and
Ward CW.
Functional studies in 3T3-L1 cells support a role for SNARE proteins in insulin stimulation of GLUT4 translocation.
Biochem J
324:
217-224,
1997[ISI][Medline].
66.
Macaulay, SL,
Rea S,
Gough KH,
Ward CW,
and
James DE.
Botulinum E toxin light chain does not cleave SNAP-23 and only partially impairs insulin stimulation of GLUT4 translocation in 3T3-L1 adipocytes.
Biochem Biophys Res Commun
237:
388-393,
1997[ISI][Medline].
67.
Maier, VH,
Melvin DR,
Lister CA,
Chapman H,
Gould GW,
and
Murphy GJ.
v- and t-SNARE protein expression in models of insulin resistance.
Diabetes
49:
618-625,
2000[Abstract].
68.
Martin, LB,
Shewan A,
Millar CA,
Gould GW,
and
James DE.
Vesicle-associated membrane protein 2 plays a specific role in the insulin-dependent trafficking of the facilitative glucose transporter GLUT4 in 3T3-L1 adipocytes.
J Biol Chem
273:
1444-1452,
1998
69.
Martin, S,
Slot JW,
and
James DE.
GLUT4 trafficking in insulin-sensitive cells. A morphological review.
Cell Biochem Biophys
30:
89-113,
1999[Medline].
70.
McBride, HM,
Rybin V,
Murphy C,
Giner A,
Teasdale R,
and
Zerial M.
Oligomeric complexes link Rab5 effectors with NSF and drive membrane fusion via interactions between EEA1 and syntaxin 13.
Cell
98:
377-386,
1999[ISI][Medline].
71.
Mills, IG,
Jones AT,
and
Clague MJ.
Regulation of endosome fusion.
Mol Membr Biol
16:
73-79,
1999[ISI][Medline].
72.
Min, J,
Okada S,
Kanzaki M,
Elmendorf JS,
Coker KJ,
Ceresa BP,
Syu LJ,
Noda Y,
Saltiel AR,
and
Pessin JE.
Synip: a novel insulin-regulated syntaxin 4-binding protein mediating GLUT4 translocation in adipocytes.
Mol Cell
3:
751-760,
1999[ISI][Medline].
73.
Nickel, W,
Weber T,
McNew JA,
Parlati F,
Sollner TH,
and
Rothman JE.
Content mixing and membrane integrity during membrane fusion driven by pairing of isolated v-SNAREs and t-SNAREs.
Proc Natl Acad Sci USA
96:
12571-12576,
1999
74.
Nishimura, Y,
Hayashi M,
Inada H,
and
Tanaka T.
Molecular cloning and characterization of mammalian homologues of vesicle-associated membrane protein-associated (VAMP-associated) proteins.
Biochem Biophys Res Commun
254:
21-26,
1999[ISI][Medline].
75.
Olson, AL,
Knight JB,
and
Pessin JE.
Syntaxin 4, VAMP2, and/or VAMP3/cellubrevin are functional target membrane and vesicle SNAP receptors for insulin-stimulated GLUT4 translocation in adipocytes.
Mol Cell Biol
17:
2425-2435,
1997[Abstract].
76.
Omata, W,
Shibata H,
Li L,
Takata K,
and
Kojima I.
Actin filaments play a critical role in insulin-induced exocytotic recruitment but not in endocytosis of GLUT4 in isolated rat adipocytes.
Biochem J
346:
321-328,
2000[ISI][Medline].
77.
Oyler, GA,
Higgins GA,
Hart RA,
Battenberg E,
Billingsley M,
Bloom FE,
and
Wilson MC.
The identification of a novel synaptosomal-associated protein, SNAP-25, differentially expressed by neuronal subpopulations.
J Cell Biol
109:
3039-3052,
1989[Abstract].
78.
Patki, V,
Virbasius J,
Lane WS,
Toh BH,
Shpetner HS,
and
Corvera S.
Identification of an early endosomal protein regulated by phosphatidylinositol 3-kinase.
Proc Natl Acad Sci USA
94:
7326-7330,
1997
79.
Peränen, J,
Auvinen P,
Virta H,
Wepf R,
and
Simons K.
Rab8 promotes polarized membrane transport through reorganization of actin and microtubules in fibroblasts.
J Cell Biol
135:
153-167,
1996[Abstract].
80.
Ploug, T,
van Deurs B,
Ai H,
Cushman SW,
and
Ralston E.
Analysis of GLUT4 distribution in whole skeletal muscle fibers: identification of distinct storage compartments that are recruited by insulin and muscle contractions.
J Cell Biol
142:
1429-1446,
1998
81.
Quinones, B,
Riento K,
Olkkonen VM,
Hardy S,
and
Bennett MK.
Syntaxin 2 splice variants exhibit differential expression patterns, biochemical properties and subcellular localizations.
J Cell Sci
112:
4291-4304,
1999
82.
Ralston, E,
Beushausen S,
and
Ploug T.
Expression of the synaptic vesicle proteins VAMPs/synaptobrevins 1 and 2 in non-neural tissues.
J Biol Chem
269:
15403-15406,
1994
83.
Ramlal, T,
Sarabia V,
Bilan PJ,
and
Klip A.
Insulin-mediated translocation of glucose transporters from intracellular membranes to plasma membranes: sole mechanism of stimulation of glucose transport in L6 muscle cells.
Biochem Biophys Res Commun
157:
1329-1335,
1988[ISI][Medline].
84.
Ramlal T, Tong P, Lam T, Somwar R, Charron M, and Klip A. The
GLUT4 compartments of skeletal muscle. In: Frontiers in Animal
Diabetes Research, edited by Zeirath JE and Shafrir E. London:
Smith-Gordon/Nishimura. In press.
85.
Randhawa, V,
Daneman N,
Regazzi R,
Bilan P,
and
Klip A.
VAMP-2, but not cellubrevin, plays a critical role in insulin-stimulated GLUT4 translocation in L6 myoblasts.
Mol Biol Cell
11:
2403-2417,
2000
86.
Ravichandran, V,
Chawla A,
and
Roche PA.
Identification of a novel syntaxin- and synaptobrevin/VAMP-binding protein, SNAP-23, expressed in non-neuronal tissues.
J Biol Chem
271:
13300-13303,
1996
87.
Rea, S,
Martin LB,
McIntosh S,
Macaulay SL,
Ramsdale T,
Baldini G,
and
James DE.
Syndet, an adipocyte target SNARE involved in the insulin-induced translocation of GLUT4 to the cell surface.
J Biol Chem
273:
18784-18792,
1998
88.
Ridley, AJ,
Paterson HF,
Johnston CL,
Diekmann D,
and
Hall A.
The small GTP-binding protein rac regulates growth factor-induced membrane ruffling.
Cell
70:
401-410,
1992[ISI][Medline].
89.
Scales, SJ,
Chen YA,
You BY,
Patel SM,
Doung YC,
and
Scheller RH.
SNAREs contribute to the specificity of membrane fusion.
Neuron
26:
457-464,
2000[ISI][Medline].
90.
Schiavo, G,
Shone CC,
Bennett MK,
Scheller RH,
and
Montecucco C.
Botulinum neurotoxin C cleaves a single Lys-Ala bond within the carboxyl-terminal region of syntaxins.
J Biol Chem
270:
10566-10570,
1995
91.
Schimmöller, F,
Simon I,
and
Pfeffer SR.
Rab GTPases, directors of vesicle docking.
J Biol Chem
273:
22161-22164,
1998
92.
Shibata, H,
Omata W,
and
Kojima I.
Insulin stimulates guanine nucleotide exchange on Rab4 via a wortmannin-sensitive signaling pathway in rat adipocytes.
J Biol Chem
272:
14542-14546,
1997
93.
Shibata, H,
Omata W,
Suzuki Y,
Tanaka S,
and
Kojima I.
A synthetic peptide corresponding to the Rab4 hypervariable carboxyl-terminal domain inhibits insulin action on glucose transport in rat adipocytes.
J Biol Chem
271:
9704-9709,
1996
94.
Skehel, PA,
Martin KC,
Kandel ER,
and
Bartsch D.
A VAMP-binding protein from Aplysia required for neurotransmitter release.
Science
269:
1580-1583,
1995[ISI][Medline].
95.
Söllner, T,
Whiteheart SW,
Brunner M,
Erdjument-Bromage H,
Geromanos S,
Tempst P,
and
Rothman JE.
SNAP receptors implicated in vesicle targeting and fusion.
Nature
362:
318-324,
1993[ISI][Medline].
96.
St-Denis, JF,
Cabaniols JP,
Cushman SW,
and
Roche PA.
SNAP-23 participates in SNARE complex assembly in rat adipose cells.
Biochem J
338:
709-715,
1999[ISI][Medline].
97.
Sumitani, S,
Ramlal T,
Liu Z,
and
Klip A.
Expression of syntaxin-4 in rat skeletal muscle and rat skeletal muscle cells in culture.
Biochem Biophys Res Commun
213:
462-468,
1995[ISI][Medline].
98.
Sutton, RB,
Fasshauer D,
Jahn R,
and
Brunger AT.
Crystal structure of a SNARE complex involved in synaptic exocytosis at 2.4 Å resolution.
Nature
395:
347-353,
1998[ISI][Medline].
99.
Tamori, Y,
Hashiramoto M,
Araki S,
Kamata Y,
Takahashi M,
Kozaki S,
and
Kasuga M.
Cleavage of vesicle-associated membrane protein (VAMP)-2 and cellubrevin on GLUT-4-containing vesicles inhibits the translocation of GLUT4 in 3T3-L1 adipocytes.
Biochem Biophys Res Commun
220:
740-745,
1996[ISI][Medline].
100.
Tamori, Y,
Kawanishi M,
Niki T,
Shinoda H,
Araki S,
Okazawa H,
and
Kasuga M.
Inhibition of insulin-induced GLUT4 translocation by munc18c through interaction with syntaxin4 in 3T3-L1 adipocytes.
J Biol Chem
273:
19740-19746,
1998
101.
Tellam, JT,
Macaulay SL,
McIntosh S,
Hewish DR,
Ward CW,
and
James DE.
Characterization of Munc-18c and syntaxin-4 in 3T3-L1 adipocytes. Putative role in insulin-dependent movement of GLUT-4.
J Biol Chem
272:
6179-6186,
1997
102.
Tellam, JT,
McIntosh S,
and
James DE.
Molecular identification of two novel Munc-18 isoforms expressed in non-neuronal tissues.
J Biol Chem
270:
5857-5863,
1995
103.
Thurmond, DC,
Ceresa BP,
Okada S,
Elmendorf JS,
Coker K,
and
Pessin JE.
Regulation of insulin-stimulated GLUT4 translocation by Munc18c in 3T3L1 adipocytes.
J Biol Chem
273:
33876-33883,
1998
104.
Thurmond, DC,
Kanzaki M,
Khan AH,
and
Pessin JE.
Munc18c function is required for insulin-stimulated plasma membrane fusion of GLUT4 and insulin-responsive amino peptidase storage vesicles.
Mol Cell Biol
20:
379-388,
2000
105.
Timmers, KI,
Clark AE,
Omatsu-Kanbe M,
Whiteheart SW,
Bennett MK,
Holman GD,
and
Cushman SW.
Identification of SNAP receptors in rat adipose cell membrane fractions and in SNARE complexes co-immunoprecipitated with epitope-tagged N-ethylmaleimide-sensitive fusion protein.
Biochem J
320:
429-436,
1996[ISI][Medline].
106.
Tong, P,
Khayat Z,
Ueyama A,
Ackerley C,
and
Klip A.
Insulin-induced actin filament remodeling is required for the translocation of glucose transporters in L6 skeletal muscle cells.
Diabetes
49, Suppl 1:
1027P,
2000.
107.
Touchot, N,
Chardin P,
and
Tavitian A.
Four additional members of the ras gene superfamily isolated by an oligonucleotide strategy: molecular cloning of YPT-related cDNAs from a rat brain library.
Proc Natl Acad Sci USA
84:
8210-8214,
1987[Abstract].
108.
Trimble, WS,
Cowan DM,
and
Scheller RH.
VAMP-1: a synaptic vesicle-associated integral membrane protein.
Proc Natl Acad Sci USA
85:
4538-4542,
1988[Abstract].
109.
Tsakiridis, T,
Vranic M,
and
Klip A.
Disassembly of the actin network inhibits insulin-dependent stimulation of glucose transport and prevents recruitment of glucose transporters to the plasma membrane.
J Biol Chem
269:
29934-29942,
1994
110.
Tsujimoto, S,
Pelto-Huikko M,
Aitola M,
Meister B,
Vik-Mo EO,
Davanger S,
Scheller RH,
and
Bean AJ.
The cellular and developmental expression of hrs-2 in rat.
Eur J Neurosci
11:
3047-3063,
1999[ISI][Medline].
111.
Uphues, I,
Kolter T,
Goud B,
and
Eckel J.
Insulin-induced translocation of the glucose transporter GLUT4 in cardiac muscle: studies on the role of small-molecular-mass GTP-binding proteins.
Biochem J
301:
177-182,
1994[ISI][Medline].
112.
Valentijn, JA,
Valentijn K,
Pastore LM,
and
Jamieson JD.
Actin coating of secretory granules during regulated exocytosis correlates with the release of rab3D.
Proc Natl Acad Sci USA
97:
1091-1095,
2000
113.
Valentijn, KM,
Gumkowski FD,
and
Jamison JD.
The subapical actin cytoskeleton regulates secretion and membrane retrieval in pancreatic acinar cells.
J Cell Sci
112:
81-96,
1999
114.
Vannucci, SJ,
Nishimura H,
Satoh S,
Cushman SW,
Holman GD,
and
Simpson IA.
Cell surface accessibility of GLUT4 glucose transporters in insulin-stimulated rat adipose cells.
Biochem J
288:
325-330,
1992[ISI][Medline].
115.
Veit, M.
Palmitoylation of the 25-kDa synaptosomal protein (SNAP-25) in vitro occurs in the absence of an enzyme, but is stimulated by binding to syntaxin.
Biochem J
345:
145-151,
2000[ISI][Medline].
116.
Vogel, K,
Cabaniols JP,
and
Roche PA.
Targeting of SNAP-25 to membranes is mediated by its association with the target SNARE syntaxin.
J Biol Chem
275:
2959-2965,
2000
117.
Volchuk, A,
Mitsumoto Y,
He L,
Liu Z,
Habermann E,
Trimble W,
and
Klip A.
Expression of vesicle-associated membrane protein 2 (VAMP-2)/synaptobrevin II and cellubrevin in rat skeletal muscle and in a muscle cell line.
Biochem J
304:
139-145,
1994[ISI][Medline].
118.
Volchuk, A,
Sargeant R,
Sumitani S,
Liu Z,
He L,
and
Klip A.
Cellubrevin is a resident protein of insulin-sensitive GLUT4 glucose transporter vesicles in 3T3-L1 adipocytes.
J Biol Chem
270:
8233-8240,
1995
119.
Volchuk, A,
Wang QH,
Ewart HS,
Liu Z,
He LJ,
Bennett MK,
and
Klip A.
Syntaxin 4 in 3T3-L1 adipocytes: Regulation by insulin and participation in insulin-dependent glucose transport.
Mol Biol Cell
7:
1075-1082,
1996[Abstract].
120.
Vollenweider, P,
Martin SS,
Haruta T,
Morris AJ,
Nelson JG,
Cormont N,
Le Marchand-Brustel Y,
Rose DW,
and
Olefsky JM.
The small guanosine triphosphate-binding protein Rab4 is involved in insulin-induced GLUT4 translocation and actin filament rearrangement in 3T3-L1 cells.
Endocrinology
138:
4941-4949,
1997
121.
Walch-Solimena, C,
Blasi J,
Edelmann L,
Chapman ER,
Fischer von Mollard G,
and
Jahn R.
The t-SNAREs syntaxin 1 and SNAP-25 are present on organelles that participate in synaptic vesicle recycling.
J Cell Biol
128:
637-645,
1995[Abstract].
122.
Wang, G,
Witkin JW,
Hao G,
Bankaitis VA,
Scherer PE,
and
Baldini G.
Syndet is a novel SNAP-25 related protein expressed in many tissues.
J Cell Sci
110:
505-513,
1997
123.
Wang, Q,
Somwar R,
Bilan PJ,
Liu Z,
Jin J,
Woodgett JR,
and
Klip A.
Protein kinase B/Akt participates in GLUT4 translocation by insulin in L6 myoblasts.
Mol Cell Biol
19:
4008-4018,
1999
124.
Weber, T,
Zemelman BV,
McNew JA,
Westermann B,
Gmachl M,
Parlati F,
Söllner TH,
and
Rothman JE.
SNAREpins: minimal machinery for membrane fusion.
Cell
92:
759-772,
1998[ISI][Medline].
125.
Weidman, PJ,
Melancon P,
Block MR,
and
Rothman JE.
Binding of an N-ethylmaleimide-sensitive fusion protein to Golgi membranes requires both a soluble protein(s) and an integral membrane receptor.
J Cell Biol
108:
1589-1596,
1989[Abstract].
126.
Weir, ML,
Klip A,
and
Trimble WS.
Identification of a human homologue of the vesicle-associated membrane protein (VAMP)-associated protein of 33 kDa (VAP-33): a broadly expressed protein that binds to VAMP.
Biochem J
333:
247-251,
1998[ISI][Medline].
127.
Wong, PPC,
Daneman N,
Volchuk A,
Lassam N,
Wilson MC,
Klip A,
and
Trimble WS.
Tissue distribution of SNAP-23 and its subcellular localization in 3T3-L1 cells.
Biochem Biophys Res Commun
230:
64-68,
1997[ISI][Medline].
128.
Yang, B,
Gonzalez L, Jr,
Prekeris R,
Steegmaier M,
Advani RJ,
and
Scheller RH.
SNARE interactions are not selective. Implications for membrane fusion specificity.
J Biol Chem
274:
5649-5653,
1999
129.
Yu, RC,
Jahn R,
and
Brunger AT.
NSF N-terminal domain crystal structure: models of NSF function.
Mol Cell
4:
97-107,
1999[ISI][Medline].
130.
Zierath, JR,
He L,
Gumà A,
Wahlström EO,
Klip A,
and
Wallberg-Henriksson H.
Insulin action on glucose transport and plasma membrane GLUT4 content in skeletal muscle from patients with NIDDM.
Diabetologia
39:
1180-1189,
1996[ISI][Medline].