mechanism and regulation of glut-4 vesicle fusion

8/8/2019 Mechanism and Regulation of GLUT-4 Vesicle Fusion

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279:877-890, 2000.Am J Physiol Cell PhysiolLeonard J. Foster and Amira Klipmuscle and fat cellsMechanism and regulation of GLUT-4 vesicle fusion in

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invited review

Mechanism and regulation of GLUT-4 vesicle fusionin muscle and fat cells

LEONARD J. FOSTER AND AMIRA KLIPCell Biology Programme, The Hospital for Sick Children, Toronto, Ontario M5G 1X8; and

Department of Biochemistry, University of Toronto, Toronto, Ontario, Canada M5S 1A8

Foster, Leonard J., and Amira Klip. Mechanism and regulation ofGLUT-4 vesicle fusion in muscle and fat cells. Am J Physiol Cell Physiol279: C877C890, 2000.Twenty years ago it was shown that recruit-ment of glucose transporters from an internal membrane compartment

to the plasma membrane led to increased glucose uptake into fat andmuscle cells stimulated by insulin. The final step of this process is thefusion of glucose transporter 4 (GLUT-4)-containing vesicles with theplasma membrane. The identification of a neuronal soluble N-ethylma-leimide-sensitive factor attachment protein receptor (SNARE) complexas a requirement for synaptic vesicle-plasma membrane fusion led to thesearch for homologous complexes outside the nervous system. Indeed,isoforms of the neuronal SNAREs were identified in muscle and fat cellsand were shown to be required for GLUT-4 incorporation into the cellmembrane. In addition, proteins that bind to nonneuronal SNAREs werecloned and proposed to regulate vesicle fusion. We have summarized themolecular mechanisms leading to membrane fusion in nonneuronalsystems, focusing on the role of SNAREs and accessory proteins(Munc18c, synip, Rab4, and VAP-33) in incorporation of GLUT-4 into theplasma membrane. Potential modes of regulation of this process are

discussed, including SNARE phosphorylation and interaction with thecytoskeleton.

vesicle traffic; solubleN-ethylmaleimide-sensitive factor attachment pro-tein receptor; syntaxin 4; 23-kDa synaptosome-associated protein-likeprotein; vesicle-associated membrane protein 2

DISCOVERY OF PROTEINS PARTICIPATING IN

VESICLE FUSION

Vesicle-membrane fusion is a fundamental cellularprocess that occurs at the final step of protein export tomost organelles and secretion of proteins and smallermolecules. Seminal work from Rothman and colleagues

(12, 20, 37, 125) in the late 1980s identified a pair ofsoluble proteins that could bind to the fusing mem-branes and were required for successful fusion of Golgi

vesicles with acceptor Golgi stacks. These proteinswere termed NSF (N-ethylmaleimide-sensitive factor)and SNAP (soluble NSF attachment protein) on thebasis of the sensitivity of the former to N-ethylmale-

imide and the ability of both proteins to bind to eachother. Later, four membrane proteins from brain extracts were found to act as receptors for NSF andSNAP 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 localizationin the presynaptic bouton, these proteins were classified as vesicle (or v-) SNAREs (e.g., VAMP-2) andtarget (or t-) SNAREs (e.g., syntaxin and SNAP-25(Table 1). VAMPs and syntaxins are characterized by a

very short extracellularly/luminally directed COOHterminus, a single transmembrane domain, and a longcytoplasmic NH2-terminal region encompassing twocoiled-coil domains (Fig. 1). In contrast, SNAP-25 doesnot have transmembrane domains but presents twocoiled-coil domains flanking a cluster of cysteine resi

Address for reprint requests and other correspondence: A. Klip,Cell Biology Programme, The Hospital for Sick Children, Toronto,Ontario, Canada M5G 1X8 (E-mail: [email protected]).

Am J Physiol Cell Physio279: C877C890, 2000

0363-6143/00 $5.00 Copyright 2000 the American Physiological Societyhttp://www.ajpcell.org C877


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dues that are highly susceptible to palmitoylation (Fig.

1). The three proteins interact with one anotherthrough their coiled-coil domains, and it is nowthought that the interaction of SNAP-25 with syntaxinis more relevant to its membrane localization than isits palmitoylation (115, 116).

It was originally proposed that the SNARE proteinswould form a link between vesicle and target mem-branes as a step preceding fusion (95) and that fusionwould be driven by the energy released from ATPhydrolyzed by bound NSF (95). Furthermore, giventhat individual SNARE isoforms were found to havedistinct tissular, cellular, and organellar specificity, itwas proposed that the SNAREs would dictate vesicletargeting specificity (95). This hypothesis is supported

by very recent work showing that only certain SNAREisoforms are able to recover disrupted norepinephrinerelease from cracked PC12 cells (89). It is now clearthat syntaxin and SNAP-25 also populate synaptic

vesicles and that VAMP is also found in target mem-branes (121). This has led to the suggestion that cis-complexes of v- and t-SNARE may occur within thesame membrane, preventing the individual compo-nents from engaging in trans-interactions with theopposite membrane. The action of NSF and SNAP isto dissociate the cis-complexes using the energy re-leased by ATP hydrolysis (Fig. 2) (7, 21). The final

fusion step depends on SNARE protein integrity butappears to be independent of ATP hydrolysis, suggesting that NSF is not involved at this stage (7, 21). Astructure-function model of fusion has been proposedwhereby 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 bundlenow described at atomic resolution (98). The free en-

ergy released by the formation of this exceptionallystable complex is thought to be the source of the energyused to fuse the two lipid bilayers (40). The elucidationof the crystal structure of the SNARE complex led to analternative classification of SNAREs into Q- orR-SNAREs, based on the presence of either a glutamine (Q) or arginine (R) residue in the center of theSNARE complex (29).

Fig. 1. Common domain structure of soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins. A: syntaxins typically contain 3 coiled-coil domains. The most COOH terminal (juxtamembrane) of these domains contains the conservedglutamine (Q) residue and participates in the SNARE complex. Bothvesicle-associated membrane proteins (VAMPs) and syntaxins havean extreme COOH-terminal transmembrane domain that protrudethrough the membrane, leaving the COOH terminus extracellular(luminal). Similar to syntaxins, VAMPs also contain a juxtamembrane coiled-coil domain that participates in the SNARE complexbut VAMP coiled-coil domains typically contain a conserved arginine(R) residue. SNAP-25 and its homologs contain 2 coiled-coil domains1 at either end of the molecule, and both domains contain conservedglutamine residues. In addition, these molecules have multiple cysteine residues (cccc) in the middle of the molecule that can bepalmitoylated to enhance the interaction of SNAP-25 with membranes.B: the 3 SNARE proteins condense into a complex containing4 -helices, 2 contributed by SNAP-25 and 1 each by VAMP andsyntaxin. The helices align in a parallel fashion, presumably alsoparallel to the plane of the membranes. The implication is that theflexible linker between the 2 coiled-coil domains of SNAP-25 mustloop back around the complex to allow both domains to align in aparallel fashion. N, NH2 terminus; C, COOH terminus.

Table 1. Nonneuronal SNAREs and interactingproteins

Protein DescriptionKey

Reference

NSF Soluble ATPase involved in fusion,sensitive to NEM

12

SNAP Soluble protein involved in fusion,binds NSF

20

Syntaxins t-SNARE, enriched on targetmembranes, many isoforms

10

SNAP-23 t-SNARE, enriched on targetmembranes, SNAP-25 homolog,three isoforms

86

VAMPs v-SNARE, enriched on vesiclemembranes, many isoforms

108

VAP-33 VAMP-associated protein, bindsVAMP via transmembranedomain

94

Pantophysin Synaptophysin homolog, bindsVAMPs

38

Synip Syntaxin 4 interacting protein,soluble

72

Munc18 Syntaxin-binding proteins, soluble,three isoforms

102

Rabs Low-molecular-weight GTPasesinvolved in vesicle traffic, manyisoforms

107

Hrs-2 Soluble protein predicted to beinvolved in vesicle traffic

9

SNAK Serine/threonine kinase showingsome substrate specificity forSNAP-23

14

NSF, N-ethylmaleimide (NEM)-sensitive factor; SNAP, solubleNSF attachment protein; t-SNARE, target soluble NSF attachmentprotein receptor (SNARE); VAMP, vesicle-associated membrane pro-tein; v-SNARE, vesicle SNARE; VAP-33, 33-kDa VAMP-associatedprotein; SNAK, SNAP-23 kinase.

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Because SNARE proteins were first identified inneuronal or neuroendocrine tissues, most of our infor-mation on these protein families stems from studies inneuronal systems. However, SNARE proteins arefound in all tissue and cell types. In the last decade,more than 9 VAMP isoforms, 19 syntaxin isoforms, and3 SNAP-25 isoforms have been described across theanimal and even the plant kingdoms (51). The conser-

vation of the basic elements of these proteins has givenrise to the tenet that these proteins must fulfill auniversal role in the mechanism of vesicle-membranefusion (for a comprehensive review, see Ref. 51).

GLUT-4 COMPARTMENTS AND THEIR SNARE

ISOFORMS

An important biological phenomenon involving vesicle-membrane fusion is the incorporation of glucosetransporters into the plasma membrane of muscle andfat cells. The glucose transporter of these tissues isGLUT-4, a 12-transmembrane domain protein thamediates vectoral transport of glucose in the directionof the glucose gradient (8). The hormone insulinstrongly promotes GLUT-4 incorporation into the celsurface, and this translocation appears to fail in insulin resistance accompanying several forms of diabetes(60, 62, 130). Because of the physiological importanceof insulin-dependent GLUT-4 translocation to the cellsurface, attempts have been made to characterize thefinal GLUT-4 vesicle fusion step, drawing from lessonslearned from neuronal synaptic transmission.

In unstimulated muscle and fat cells, the steadystate 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 theplasma membrane (47, 53). Most studies suggest thatthe intracellular compartments populated by GLUT-4include the early/sorting endosome, the recycling endosome, and a specialized vesicular compartment (4155, 56, 69). It is currently debated whether the latterdoes or does not recycle in the basal state. In rodentadipocytes, insulin promotes the externalization of thespecialized vesicles and increases the recycling oGLUT-4 from the recycling endosome to the plasmamembrane (41, 55, 56, 69). In muscle, insulin mobilizesa specialized vesicle pool, but there is no evidence thatthe recycling endosome is also mobilized (1, 2). Insteademerging studies are consistent with the possibilitythat muscle contraction mobilizes GLUT-4 from therecycling endosome in this tissue (24, 63, 80). ThusGLUT-4-containing vesicles incorporate into theplasma membrane in at least three circumstances: inthe basal (unstimulated) state, out of the recyclingendosome; in response to insulin, out of the specialized

vesicle; and in response to exercise in muscle and toinsulin in fat cells, out of the recycling endosomeOther possibilities are not discounted, such as additional mobilization of the specialized vesicular pool inresponse to exercise. This diversity of fusion eventsbegs 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 expressedin muscle and fat cells had first to be defined. Of the

VAMP family, only VAMP-2 and VAMP-3/cellubrevinhave so far been detected in muscle and fat primarytissues and corresponding cells in culture (82, 105117). Contrary to neuronal and neuroendocrine cellsmuscle and fat cells do not express syntaxin 1, butinstead express syntaxin 4 (49, 102, 119). In additionlow levels of syntaxin 2 are also detected in 3T3-L1adipocytes (119) and rat adipocytes (105), whereassmall levels of syntaxin 3 are present in rat adipocytes

Fig. 2. Hypothetical steps in vesicle docking and fusion applied tothe glucose transporter 4 (GLUT-4) system. Preformed cis-SNAREcomplexes on the plasmalemmal and vesicle membranes must firstbe dissociated by the action of the ATPase N-ethylmaleimide-sensi-tive factor (NSF) and its assistant, soluble NSF attachment protein(SNAP) (priming). The vesicle then becomes associated with theplasma membrane (tethering) through as yet unknown molecules.Rab4 may participate at this point, but tethering does not involveSNARE proteins. Once tethered, the SNAREs can trans-associate,causing the vesicle to become more tightly associated with theplasma membrane (docking). Docking then leads to formation of theclassic SNARE complex on the way to fusion of the vesicle with theplasma membrane.

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(105). Another difference between neuronal/neuroen-docrine cells and muscle and fat cells pertains to theexpression of SNAP-25. This isoforms was not found ininsulin-sensitive cells, which instead express SNAP-23(3, 122, 127).

THE PREFUSION STEPS

Synaptic vesicle fusion is the final step in a series of

events that have been described as vesicle tethering (areversible step) and vesicle docking (an irreversiblestep) (Fig. 2). Information on these steps has alsoemerged from yeast molecular genetic studies (16) andfrom in vitro endosome-endosome fusion studies (19,71). The full complement of tethering proteins has notyet been identified, but the early endosome autoanti-gen 1 (EEA1) protein may act as the tethering proteinengaged in binding endocytic vesicles to the early en-dosome (19, 70). EEA1 is found in insulin-sensitive celltypes (78), but its participation in GLUT-4 vesicletraffic has not been tested.

Once vesicles are brought into close proximity withtheir target membranes by the tethering process,SNARE proteins on opposite membranes associate andacquire the configuration required for fusion (Fig. 2,docked). The SNARE proteins in docked vesicles arethought to be in a high-energy state (40) that can bemaintained for long periods of time. Indeed, large num-bers of synaptic vesicles can be observed docked at thepresynaptic plasma membrane (51). In contrast,GLUT-4 vesicles have rarely, if at all, been foundperched at the plasma membrane of unstimulatedmuscle or fat cells.

In neurons, nerve terminal depolarization leading tocalcium influx is the penultimate trigger for neuro-transmitter exocytosis. Currently, there is no evidence

supporting a need for calcium ions in insulin-depen-dent glucose uptake mediated by GLUT-4 in 3T3-L1adipocytes, L6 myotubes, or cardiac myocytes (42, 59,61). In fact, GLUT-4 insertion into the plasma mem-brane can be observed in cells equilibrated with calci-um-free buffers by means of streptolysin O-induced cellpermeabilization (17) (Foster LJ and Klip A, unpub-lished observation).

PROTEINS PARTICIPATING IN GLUT-4 VESICLE

FUSION

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 syn-apses (26), insulin-stimulated GLUT-4 translocation infat and muscle cells (15, 117), and aquaporin-2 trans-location in renal collecting ducts (54). VAMP-2 is ex-pressed in muscle (82, 117) and fat cells (15, 118) andwas originally detected in immunoisolated GLUT-4compartments from rat fat cells (15). By subcellularfractionation of muscle and adipose cells, the protein isfound to be distributed in similar proportions in theplasma membrane and intracellular membranes (15,117, 118).

VAMP-2 is susceptible to cleavage by various clostridium neurotoxins (50). This susceptibility afforded aspecific strategy to probe the function of VAMP-2 (anda closely related isoform called VAMP-3/cellubrevin) inGLUT-4 traffic. We and others have demonstrated arequirement for VAMP-2 in insulin-stimulatedGLUT-4 translocation (17, 18, 31, 39, 65, 68, 75, 8599). Tetanus toxin and botulinum toxins B and D

introduced into rodent adipocytes by electroporationsingle-cell microinjection, chemical permeabilization(using streptolysin O toxin), or natural, toxin-mediateduptake (17, 18, 31, 39, 65, 99) reduced by more thanone-half the insulin-stimulated GLUT-4 incorporationinto the cell surface (Table 2). In addition, introductionof antibodies raised against various regions of VAMP-2as well as peptides representing different segments of

VAMP-2 also diminished the insulin-dependent arrivaof GLUT-4 at the plasma membrane of rodent adipocytes (17, 65, 68, 75) (Table 2).

Recent work on the function of VAMPs in GLUT-4traffic has focused on resolving whether VAMP-2 or

VAMP-3/cellubrevin is the primary v-SNARE involved ininsulin-stimulated GLUT-4 translocation. It has beensuggested that VAMP-2 is the v-SNARE important fortranslocation of GLUT-4 from the insulin-sensitive compartment, because the cytosolic domain of VAMP-2, butnot VAMP-3/cellubrevin or VAMP-1, reduced insulinstimulated GLUT-4 translocation by one-half when microinjected into 3T3-L1 adipocytes (68). In additiontransfection of tetanus toxin light chain into L6 musclecells in culture resulted in 70% inhibition of insulindependent GLUT-4 arrival at the cell surface (85). Basalevels of cell surface GLUT-4 were minimally affectedCotransfection of tetanus toxin-insensitive mutants o

VAMP-2, but not VAMP-3, rescued the inhibition (85)These results indicate that VAMP-2, but not VAMP-3, isinvolved in insulin-stimulated GLUT-4 translocation andthat neither protein participates in GLUT-4 sorting tothe plasma membrane in the basal state.

Syntaxin 4

Syntaxin 4 is expressed in muscle and fat cellswhere it is largely, but not exclusively, located at theplasma membrane (97, 105, 119). In fact, GLUT-4

vesicles contain syntaxin 4 that cannot be explained bycontamination from plasma membranes (119). Unlikesyntaxins 1 through 3, syntaxin 4 is not susceptible tocleavage by botulinum toxin C1 (90). For this reasonstudies on the functional role of syntaxin 4 in GLUT-4translocation have required the use of antibodies andpeptides to perturb the function of syntaxin 4. Microinjection (65, 75, 101), chemical permeabilization (17119), and adenoviral overexpression (75) have beenused to introduce antibodies directed against syntaxin4 or soluble domains of the protein. In all cases, theperturbation of syntaxin 4 resulted in 50% inhibitionof insulin-stimulated glucose uptake (119) or GLUT-4translocation (17, 65, 75, 101) (Table 2).

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SNAP-23

SNAP-23 shares both sequence and structural ho-mology with SNAP-25 (86). A protein cloned from acDNA library of 3T3-L1 adipocytes, originally namedsyndet (122), was found to be the murine form ofSNAP-23 (96). By subcellular fractionation of muscleand fat cells, SNAP-23 is found almost exclusively inthe plasma membrane-enriched fraction (122, 127).Neutralizing antibodies as well as peptides encodingthe NH2 or COOH termini of SNAP-23 have beenintroduced into 3T3-L1 adipocytes by microinjection,chemical permeabilization, and adenoviral transfec-tion. All these reagents reduced the insulin-dependentarrival of GLUT-4 at the plasma membrane, although

they did not inhibit it completely (31, 34, 58, 87). Theclostridium neurotoxins Bo/NT A and E have beenuseful to probe the function of SNAP-25, but they havebeen less effective in targeting SNAP-23. Bo/NT E hasbeen shown to cleave SNAP-23 in some species, notablythe canine isoform (64). In some reports, the toxin wasable to cleave the murine SNAP-23, concomitantly re-ducing GLUT-4 translocation (31). In other studies, thetoxin was ineffective toward SNAP-23 (18, 66). Be-cause SNAP-23 is not a transmembrane protein andcan bind to native syntaxin 4, it was also possible tointroduce into cells full-length SNAP-23 to test its

function. The microinjected full-length protein en

hanced both insulin-stimulated GLUT-4 translocationand glucose uptake (34). These results suggest that theendogenous SNAP-23 may be available for SNAREcomplex formation in limiting amounts. A very recentreport (43) has defined that 3T3-L1 adipocytes haveapproximately 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 extentof availability of each of these proteins for SNAREcomplex formation is still to be determined, given thatthese proteins have several cellular partners. Exogenous SNAP-23 may enhance the rate of fusion oGLUT-4 vesicles with the plasma membrane by enhancing the formation of productive complexes withsyntaxin 4 and VAMP-2 (Table 2).

NSF and SNAP

Unlike the expanded nature of the SNARE proteinfamilies, NSF and SNAP have very few apparenthomologs. High-resolution X-ray crystal structuressuggest that NSF may engage SNAP as a lever to prythe SNARE complex apart (129) (Table 1). This wouldthen allow SNAREs to form complexes between opposing membranes that are competent for fusion. IndeedNSF and SNAP are found in rat adipocytes, and

Table 2. Effects of interfering with SNARE proteins on insulin-stimulated glucose uptakeand/or GLUT-4 translocation

Cell Type Reagent and Method of IntroductionGLUT-4

TranslocationGlucoseUptake Reference

VAMP-2

3T3-L1 Botulinum toxin D by streptolysin O toxin Decreased n.d. 173T3-L1 Botulinum toxin B by streptolysin O toxin Decreased Decreased 99

3T3-L1 Cytoplasmic domain by streptolysin O toxin Decreased n.d. 17, 683T3-L1 NH2-terminal peptide by microinjection Decreased n.d. 653T3-L1 Tetanus toxin by microinjection Decreased n.d. 653T3-L1 Botulinum toxin B by microinjection Decreased n.d. 31, 653T3-L1 Botulinum toxin B by toxin-mediated uptake n.d. Decreased 183T3-L1 Cytoplasmic domain by adenoviral transfection Decreased n.d. 75Rat adipocytes Tetanus toxin by electroporation No effect No effect 393T3-L1 NH2-terminal peptide by streptolysin O toxin Decreased n.d. 68L6 myoblasts Tetanus toxin by transfection Decreased n.d. 85

Syntaxin 4

3T3-L1 Cytoplasmic domain by streptolysin O toxin Decreased n.d. 173T3-L1 Antibody by streptolysin O toxin n.d. Decreased 1193T3-L1 Internal peptide (10622) by microinjection Decreased n.d. 653T3-L1 Cytoplasmic domain by adenoviral transfection Decreased n.d. 753T3-L1 Cytoplasmic domain by microinjection Decreased n.d. 753T3-L1 Antibody by microinjection Decreased n.d. 101

SNAP-23

3T3-L1 Botulinum toxin E by toxin-mediated uptake n.d. No effect 183T3-L1 Botulinum toxin E by microinjection No effect n.d. 663T3-L1 COOH-terminal peptide by microinjection Decreased n.d. 873T3-L1 NH2-terminal antibody by microinjection Decreased n.d. 873T3-L1 COOH-terminal antibody by microinjection Decreased n.d. 343T3-L1 Full-length protein by microinjection Increased n.d. 343T3-L1 Full-length protein by streptolysin O toxin n.d. Increased 343T3-L1 Botulinum toxin E by microinjection Decreased n.d. 313T3-L1 Full-length protein by adenoviral transfection No effect No effect 583T3-L1 NH2-terminal 202 amino acids by adenoviral transfection Decreased Decreased 583T3-L1 NH2-terminal 161 amino acids by adenoviral transfection No effect No effect 58

GLUT-4, glucose transporter 4; n.d., not determined.

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epitope-tagged versions of these proteins have beenused to immunoprecipitate SNARE complexes fromthese cells. Such complexes contained syntaxin 4,

VAMP-2, VAMP-3, and SNAP-23 (105). Transfection ofa dominant negative mutant of NSF into rat adipocytesresulted in plasma membrane levels of GLUT-4 afterinsulin treatment that were not significantly differentfrom basal, nontransfected levels (45). However, the

level of plasma membrane GLUT-4 in basal cells ex-pressing the dominant negative NSF was also loweredsignificantly (45).

DO SNARES SUFFICE TO CAUSE GLUT-4 VESICLE

FUSION?

The three SNAREs expressed in muscle and fat cellsappear to be essential for a significant fraction of theinsulin-stimulated GLUT-4 arrival at the cell surface.However, in all the experiments discussed above, in-terfering with VAMP-2, syntaxin 4, or SNAP-23 onlypartly inhibited insulin-stimulated GLUT-4 transloca-tion, and the basal levels of plasma membrane GLUT-4were not altered even after long time periods in thepresence of the perturbing agent. This latter observa-tion is especially surprising given that GLUT-4 isknown to cycle dynamically to and from the membranein the absence of insulin. These observations suggestthat either different SNARE isoforms or other proteinsmediate these two fusion events. Only one other VAMPhas been detected in the relevant membranes of muscleand fat cells, VAMP-3/cellubrevin. However, completehydrolysis of this protein by botulinum or tetanustoxin, or interference by peptides emulating NH2-ter-minal sequences of VAMP-3/cellubrevin, failed to affecteither basal or insulin-mediated GLUT-4 arrival at thecell surface (68, 75, 85), whereas the analogous domain

of VAMP-2 effectively inhibited one-half of the insulinaction (68). It is conceivable that muscle and fat cellsexpress other, toxin-insensitive VAMPs, which couldpotentially mediate the fusion events that are not ac-counted for by VAMP-2. Similarly, it is conceivablethat syntaxin 2 or 3 could mediate fusion events be-cause they each bind to VAMP-2 (28, 81).

The studies listed above support the notion thatVAMP-2, syntaxin 4, and SNAP-23 are required for theincorporation of GLUT-4-containing vesicles into theplasma membrane. These proteins may participate inthe actual membrane fusion step, by analogy to the

fusogen role assigned to their neuronal counterparts(51). Indeed, purified, bacterially expressed SNAP-25syntaxin 1, and VAMP-2 reconstituted into syntheticproteoliposomes can mediate fusion of these liposomes(73, 124). Fusion of a single vesicle with its targetmembrane likely requires the formation of more thanone SNARE complex, and there is a suggestion that aring of SNARE complexes aligns around the fusionpore (124). Current experiments have not been able todetermine the number of complexes required for fusionof one vesicle. This will likely require detailed microcalorimetry experiments measuring the free energyreleased during complex formation.

The importance of the formation of a neuronaSNARE complex for synaptic vesicle fusion suggeststhat a high-affinity complex may also form between

VAMP-2, syntaxin 4, and SNAP-23 in the process oGLUT-4 vesicle fusion. This possibility has been addressed experimentally, yielding somewhat surprisingresults (Table 3). Although there is general agreementthat a complex does form comprising SNAP-23, syn

taxin 4, and VAMP-2, the biochemical properties osuch a complex appear to differ from those of the

VAMP-2/syntaxin 1/SNAP-25 complex. These differences depend on the experimental design includingimportantly, whether the proteins used are recombinant forms produced in bacteria or endogenous proteins isolated from mammalian cell systems. One of thehallmarks of the neuronal SNARE complex involvingSNAP-25, syntaxin 1, and VAMP-2 is its ability toresist denaturation by ionic detergents such as sodiumdodecyl sulfate (SDS). Whereas an SDS-resistant complex containing VAMP-2, SNAP-23, and syntaxin 4 in0.5% SDS was detected using surface plasmon reso

nance (87), such a complex could not be detected bySDS-PAGE or circular dichroism using samples in 2%SDS (35, 128). A second distinguishing feature of theneuronal SNARE complex is that SNAP-25 enhancesthe binding of VAMP-2 to syntaxin 1. In contrast, inglutathione S-transferase-pulldown experiments, wefound no evidence for cooperative binding betweenSNAP-23, VAMP-2, and syntaxin 4 (35); however, acooperative effect of SNAP-23 on complex formationwas reported when all three proteins were overexpressed in COS cells (58).

Table 3. Reported SNARE complexes containing SNAP-23, VAMP-2, and syntaxin 4

System Detection Method CooperativitySDS

Resistance Reference

Immunopurified from rat adipocyte cell lysates SDS-PAGE n.d. n.d. 105Recombinant cytosolic domains, affinity purified SDS-PAGE None No 35Recombinant cytosolic domains, size-exclusion purified,

SDS-PAGE SDS-PAGE, CD n.d. No 128Recombinant cytosolic domains, affinity purified SPR n.d. Yes 87Immunopurified from rat adipocyte cell lysates SDS-PAGE Enhanced by SNAP n.d. 96Immunopurified from 3T3-L1 and COS cell lysates SDS-PAGE Yes, in COS cells n.d. 58Neuronal complex of syntaxin 1, VAMP-2, and SNAP-25 SDS-PAGE, CD, SPR Enhanced by SNAP-25 Yes 51

CD, circular dichroism; SPR, surface plasmon resonance.

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clone similar genes from a 3T3-L1 cDNA library. Oneof the proteins cloned by this method was Munc18c,which interacts specifically with syntaxins 2 and 4 butnot syntaxins 1 or 3 (100, 102). Munc18c inhibits the

binding of syntaxin 4 to VAMP-2 (101, 103) andSNAP-23 (3). Insulin causes the dissociation of aMunc18c/syntaxin 4 complex (103). A prediction of thisobservation is that once insulin causes the dissociation

Table 4. Phosphorylation of SNAREs and ancillary proteins

Protein SystemHormone

RegulationLevel/

StoichiometryKinase

ResponsibleEffect of

Phosphorylation Referenc

Syntaxin 4 Immune complex from3T3-L1 adipocytes

None with INSor IPT

Low Unknown Unknown

Syntaxin 4 Immune complex fromHeLa cells

n.d. Low Unknown, notSNAK

Unknown 14

Syntaxin 4 Recombinant protein/

purified kinase

n.a. 3.9 mol/mol Protein

kinase A

Inhibits binding to

SNAP-23

35

Syntaxin 4 Recombinant protein/recombinant kinase

n.a. 0.81 mol/mol Casein kinaseII

Unknown 35

Syntaxin 4 Recombinant protein/purified kinase

n.a. 0.05 mol/mol Proteinkinase C

Unknown 35

Syntaxin 4 Recombinant protein/recombinant kinase

n.a. Low SNAK Unknown 14

SNAP-23 Recombinant protein/purified kinase

n.a. 0.01 mol/mol Proteinkinase C

Unknown 35

SNAP-23 Immune complex from3T3-L1 adipocytes

None with INSor IPT

Low Unknown Unknown

SNAP-23 Recombinant protein/recombinant kinase

n.a. High SNAK Enhances binding tosyntaxin 4

14

VAMP-2 Immune complex from3T3-L1 adipocytes

None with INS Low Unknown Unknown

Pantophysin Immune complex from3T3-L1 adipocytes

None with INS High Unknown Unknown 13

77-kDaunknown

Bound to pantophysinimmune complex INS increases Unknown Unknown Unknown 13

Ins, insulin; Iso, isoproterenol; n.a., not applicable. *Foster L and Klip A, unpublished observations.

Table 5. Effects of interfering with SNARE-binding proteins on insulin-stimulated glucose uptakeor GLUT-4 translocation

Cell Type Reagent and Method GLUT-4Translocation GlucoseUptake Referenc

Munc18c

3T3-L1 Full-length protein by adenoviral transfection Decreased n.d. 1033T3-L1 Full-length protein by adenoviral transfection Decreased Decreased 1003T3-L1 Syntaxin 4-binding peptide by microinjection Decreased n.d. 104

Synip

3T3-L1 COOH-terminal half by adenoviraltransfection/microinjection

Decreased Decreased 72

3T3-L1 Full-length protein by adenoviral transfection/microinjection No effect No effect 723T3-L1 NH2-terminus half by adenoviral transfection/microinjection No effect No effect 72

Rab4

Rat adipocytes Peptide (191210) by electroporation Decreased Decreased 93Rat adipocytes Antibody by electroporation Decreased Decreased 93

Rat adipocytes Full-length protein by transfection Decreased n.d. 22Rat adipocytes Membrane-association mutant by transfection Decreased n.d. 223T3-L1 Full-length protein by microinjection No effect n.d. 1203T3-L1 GTP-binding mutant by microinjection Decreased n.d. 1203T3-L1 GTPase mutant by microinjection No effect n.d. 1203T3-L1 Antibody by microinjection Decreased n.d. 120

VAP-33

3T3-L1 Antibody by microinjection Decreased n.d. 33L6 myoblasts Full-length protein by transfection Decreased n.d. 33

Pantophysin

3T3-L1 Antibody by microinjection Decreased n.d.

*Foster LJ, Cheatham B, and Klip A, unpublished observations.

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syntaxin 4 would be available to bind SNAP-23 and VAMP-2, leading to fusion of the vesicles with thetarget membrane. Indeed, full-length Munc18c intro-duced into 3T3-L1 adipocytes by adenoviral transfec-tion inhibited insulin-stimulated glucose uptake andGLUT-4 translocation by 50% (100, 103). However, apeptide representing the domain of Munc18c thatbinds to syntaxin 4, when microinjected into 3T3-L1

adipocytes, inhibited fusion of green fluorescent pro-tein-GLUT-4-containing vesicles with the plasmamembrane. The peptide appeared to allow GLUT-4

vesicles to dock with the plasma membrane withoutfusing with it. Given that this peptide displacesMunc18c-binding to syntaxin 4, these results may sug-gest that the displaced, endogenous Munc18c catalyzesfusion (104) (Fig. 3).

Synip. The recently cloned synip is a syntaxin 4-in-teracting protein, identified in a 3T3-L1 cDNA libraryby a yeast two-hybrid screen (72). Synip binding tosyntaxin 4 prevents VAMP-2/syntaxin 4 binding butnot SNAP-23/syntaxin 4 binding (72). As for Munc18c,the association of synip with syntaxin 4 is reduced ininsulin-stimulated cells. Insulin-sensitivity is con-ferred by the NH2-terminal half of synip, whereas theCOOH-terminal half modulates GLUT-4 translocation(72). Despite having unrelated primary sequences, sy-nip and Munc18c regulate the availability of syntaxin 4for fusion of GLUT-4 vesicles with the plasma mem-brane in response to insulin (Fig. 3). It will be inter-esting to determine whether the two proteins regulatedifferent functional pools of syntaxin 4.

SNAK. SNAK is a protein kinase identified by itsability to bind syntaxin 4 in a yeast two-hybrid assay(14). However, SNAP-23 is a better substrate of SNAKthan syntaxin 4 (14). SNAK phosphorylates SNAP-23

in vivo and in vitro, selectively phosphorylating onlySNAP-23 that is not bound to syntaxin 4. SNAK phos-

phorylation of SNAP-23 enhances t-SNARE complexassembly, 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 byinsulin. Results of in vivo phosphorylation do not sup-port any insulin-dependent phosphorylation oSNAP-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 inmuscle and fat cells, but its tyrosine phosphorylationstate is not altered in response to insulin (Yaworsky KFoster LJ, and Klip A, unpublished observations). Todate, there is no evidence for its participation as aregulator of GLUT-4 traffic.

Pantophysin. A ubiquitous homolog of the synaptic vesicle protein synaptophysin, termed pantophysinhas recently been cloned from several sources (13, 38)This protein is found on GLUT-4-containing vesiclesfrom 3T3-L1 cells and, similarly to synaptophysinbinds VAMP-2 (13). Interestingly, although pantophysin itself was not phosphorylated, a 77-kDa phosphoprotein associates with pantophysin upon treatment ofcells with insulin (13). This result suggests a potentiaregulation of pantophysin by insulin. Preliminary results from our laboratory suggest that pantophysinavailability is required for GLUT-4 vesicle fusion (Foster LJ, Cheatham B, and Klip A, unpublished observa-tions).

VAP-33. A 33-kDa VAMP-2-associating protein(VAP-33) was isolated from an Aplysia californicacDNA library through a yeast two-hybrid approach(94). A human homolog was identified soon thereafter(126). VAP-33 is a single-transmembrane domain pro

tein with the bulk of the molecule in the cytosol. Twoisoforms of VAP-33 (VAP-33A and VAP-33B) bind

Fig. 3. Hypothetical regulatory events in GLUT-4 vesicle docking and fusion. In the basal state, both Munc18c andsynip are bound to syntaxin 4, whereas SNAP-23 and GLUT-4 are associated with the actin cytoskeleton.Pantophysin, VAMP-2, and Rab4 are located on GLUT-4 vesicles. Before insulin causes the release of Rab4 fromthe vesicles, Rab4 organizes the proteins on the vesicles into conformations required for fusion. A 33-kDaVAMP-2-associating protein (VAP-33) may also be involved as a chaperone for VAMP-2. Insulin causes theformation of actin ruffling and the dissociation of Munc18c and synip from syntaxin 4. Once brought into theproximity of fusion machinery at the plasma membrane by the cytoskeleton, the GLUT-4 vesicle can dock andsubsequently fuse, exposing GLUT-4 to the extracellular milieu.

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VAMP-2 in vitro (74). We have recently shown (33) thatVAP-33 is present on immunopurified VAMP-2 vesiclesfrom L6 myotubes and 3T3-L1 adipocytes. Interest-ingly, overexpression of VAP-33A inhibited insulin-stimulated GLUT-4 translocation, and this effect wasrescued by co-overexpression of VAMP-2. In addition,anti-VAP-33 antibodies microinjected into 3T3-L1 adi-pocytes also inhibited GLUT-4 translocation (33). We

hypothesize that, as in the case of Munc18c, the levelsof VAP-33A in the cell are critical and that shifting thebalance of VAP-33A/B either above or below the criticalpoint can have adverse effects on vesicle traffic.

Rab proteins. Rab GTPases represent a family of35 proteins that relay information upon binding intheir GTP-bound form to downstream effectors. Rabsbecome membrane-associated via geranylgeranylationor farnesylation, and this posttranslational modifica-tion is required for their GTPase function, which ter-minates their function on effectors. By genetic comple-mentation, Rab proteins have been implicated in

vesicular traffic, specifically in the recognition of vesi-cles by target membranes (for review, see Ref. 91).Deletion of the yeast Rab4 (Sec4p) can be rescued byoverexpression of specific t-SNAREs, and the Rab

Ypt1p is required for v-SNARE-t-SNARE complex for-mation. An emerging model suggests that Rab proteinsdirect vesicle traffic through the recruitment of dockingfactors from the cytosol. Thus Sec4 binds to the yeastexocyst that links vesicles to bud membranes, Rab5binds to EEA1 that links endocytic vesicles to earlyendosomes, and vesicular VPs21 binds to Vac1 thatlinks to the t-SNARE Ppe12 on target vesicles (4).

To date, only Rab4 has been implicated in GLUT-4traffic by virtue of its presence on immunopurifiedGLUT-4 compartments (23, 111). Insulin stimulation

causes Rab4 geranylgeranylation, GTP loading (92),and dissociation from GLUT-4-containing endomem-branes (22, 36). Introduction of wild type or mutants ofRab4 or a peptide representing the hypervariable re-gion of Rab4 resulted in inhibition of insulin-stimu-lated GLUT-4 translocation (22, 93, 120). Interest-ingly, a link between the Rab-mediated vesicle dockingand the actin-based cytoskeleton has been established.Rabphilin, a Rab3 effector, interacts with the actin-bundling protein -actinin (57), and Rab8 promotespolarized membrane transport through reorganizationof actin filaments (79).

The Cytoskeleton

The actin cytoskeleton has been repeatedly impli-cated in exocytic events, both as a barrier separatingthe docked from stored synaptic vesicles (in essence,limiting the active zone) and as a facilitator of granuleexocytosis (11, 112). Recent studies reveal that secre-tory granules acquire a coat of actin before exocytosis(113). Actin filaments are dynamic and, in addition toseparating active zones and coating granules, theyconstitute stress fibers and cortical networks. The lat-ter form in response to growth factor stimulation in-

volving the Rho-family protein Rac (88), and in insulin-

sensitive muscle cells, they present as largesubmembranous three-dimensional structures (59109). We have recently shown that formation of corticaactin structures is required for GLUT-4 exocytosisSpecifically, the rapidly forming subcortical actin meshcontained GLUT-4 vesicles and insulin signaling molecules (59). Preventing cortical actin structure formation through transient expression of a dominant nega

tive Rac mutant abrogated externalization of GLUT-4(59). In nontransfected muscle cells, GLUT-4 is inserted into the membrane at sites of membrane rufflessupported by cortical actin structures (106). NotablySNAP-23 and syntaxin 4 appear to concentrate at sitesof contact of the actin mesh with the plasma membrane(Khayat K, Foster LJ, and Klip A, unpublished obser

vations). In adipocytes, a requirement for an organizedcytoskeleton in GLUT-4 traffic has also been demonstrated (76). It will be interesting to determinewhether GLUT-4 vesicles, once delivered by the cytoskeleton to the vicinity of the plasma membranerequire Rab4 as the tethering molecule leading toSNARE complex formation.

CONCLUSION

GLUT-4 vesicle fusion bears similarities to and differences from the fusion of synaptic vesicles with theirrespective target membranes. VAMP-2, syntaxin 4and SNAP-23, found in muscle and fat cells, form aSNARE complex that is similar but not identical to itsneuronal counterpart, constituted by VAMP-2, syntaxin 1, and SNAP-25. Two mechanisms of insulindependent incorporation of GLUT-4 vesicles into theplasma 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 isno evidence for regulation through SNARE phosphorylation. However, it is conceivable that subtle regulation may still occur by this means. In contrast, there isemerging evidence that ancillary proteins such asMunc18c, 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 theprocess of GLUT-4 vesicle fusion. Future studiesshould reveal whether the fine tuning of GLUT-4 vesicle fusion is altered in insulin-resistant states leadingto or accompanying diabetes. In this regard, two recentstudies (67, 84) report increases in muscle SNARE

protein levels in two animal models of insulin resis-tance. Both reports suggest that these increases mightbe adaptive changes attempting to overcome the defects in GLUT-4 traffic that underlie insulin resistance

We acknowledge the Juvenile Diabetes Foundation and the Medical Research Council of Canada for funding.

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