Mechanism and regulation of GLUT-4 vesicle fusion in

muscle and fat cells

Leonard J. Foster and Amira Klip
Am J Physiol Cell Physiol 279:877-890, 2000.

You might find this additional information useful...

This article cites 112 articles, 71 of which you can access free at:
http://ajpcell.physiology.org/cgi/content/full/279/4/C877#BIBL

This article has been cited by 23 other HighWire hosted articles, the first 5 are:
Exocytosis mechanisms underlying insulin release and glucose uptake: conserved roles for
Munc18c and syntaxin 4
J. L. Jewell, E. Oh and D. C. Thurmond
Am J Physiol Regulatory Integrative Comp Physiol, March 1, 2010; 298 (3): R517-R531.
[Abstract] [Full Text] [PDF]

Current views on type 2 diabetes

Y. Lin and Z. Sun

Downloaded from ajpcell.physiology.org on October 29, 2010

J. Endocrinol., January 1, 2010; 204 (1): 1-11.
[Abstract] [Full Text] [PDF]

Membrane Fatty Acid Transporters as Regulators of Lipid Metabolism: Implications for

Metabolic Disease
J. F. C. Glatz, J. J. F. P. Luiken and A. Bonen
Physiol Rev, January 1, 2010; 90 (1): 367-417.
[Abstract] [Full Text] [PDF]

Knockdown of TRPC3 with siRNA coupled to carbon nanotubes results in decreased

insulin-mediated glucose uptake in adult skeletal muscle cells
J. T. Lanner, J. D. Bruton, Y. Assefaw-Redda, Z. Andronache, S.-J. Zhang, D. Severa, Z.-B.
Zhang, W. Melzer, S.-L. Zhang, A. Katz and H. Westerblad
FASEB J, June 1, 2009; 23 (6): 1728-1738.
[Abstract] [Full Text] [PDF]

Murine CENPF interacts with syntaxin 4 in the regulation of vesicular transport

R. D. Pooley, K. L. Moynihan, V. Soukoulis, S. Reddy, R. Francis, C. Lo, L.-J. Ma and D. M.
Bader
J. Cell Sci., October 15, 2008; 121 (20): 3413-3421.
[Abstract] [Full Text] [PDF]

Medline items on this article's topics can be found at http://highwire.stanford.edu/lists/artbytopic.dtl

on the following topics:
Biochemistry .. Membrane Fusion
Biochemistry .. Glucose Transport
Biochemistry .. Glucose Uptake
Biochemistry .. Fats
Biochemistry .. Fat Cell
Cell Biology .. Vesicle Fusion
Updated information and services including high-resolution figures, can be found at:
http://ajpcell.physiology.org/cgi/content/full/279/4/C877

Additional material and information about AJP - Cell Physiology can be found at:
http://www.the-aps.org/publications/ajpcell

This information is current as of October 29, 2010 .

AJP - Cell Physiology is dedicated to innovative approaches to the study of cell and molecular physiology. It is published 12 times
a year (monthly) by the American Physiological Society, 9650 Rockville Pike, Bethesda MD 20814-3991. Copyright © 2000 by the
American Physiological Society. ISSN: 0363-6143, ESSN: 1522-1563. Visit our website at http://www.the-aps.org/.
 Am J Physiol Cell Physiol
279: C877–C890, 2000.

invited review
Mechanism and regulation of GLUT-4 vesicle fusion
in muscle and fat cells

LEONARD J. FOSTER AND AMIRA KLIP

Cell 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 of

GLUT-4 vesicle fusion in muscle and fat cells. Am J Physiol Cell Physiol

Downloaded from ajpcell.physiology.org on October 29, 2010

279: C877–C890, 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 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-ethylma-
leimide-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 pro-
tein receptor; syntaxin 4; 23-kDa synaptosome-associated protein-like
protein; vesicle-associated membrane protein 2

DISCOVERY OF PROTEINS PARTICIPATING IN
VESICLE FUSION
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 mem-
branes 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-ethylmale-
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@sickkids.on.ca).
http://www.ajpcell.org 0363-6143/00 $5.00 Copyright © 2000 the American Physiological Society
imide and the ability of both proteins to bind to each
other. Later, four membrane proteins from brain ex-
tracts 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-associ-
ated membrane protein-2) (26), syntaxins A and B (10),
and SNAP-25 (25-kDa synaptosome-associated pro-
teins) (77). On the basis of their topological localization
in the presynaptic bouton, these proteins were classi-
fied 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 resi-
C877
C878
Table 1. Nonneuronal SNAREs and interacting
proteins
Protein Description
NSF Soluble ATPase involved in fusion,
sensitive to NEM
␣SNAP Soluble protein involved in fusion,
binds NSF
Syntaxins t-SNARE, enriched on target
membranes, many isoforms
SNAP-23 t-SNARE, enriched on target
membranes, SNAP-25 homolog,
three isoforms
VAMPs v-SNARE, enriched on vesicle
membranes, many isoforms
VAP-33 VAMP-associated protein, binds
VAMP via transmembrane
domain
Pantophysin Synaptophysin homolog, binds
VAMPs
Synip Syntaxin 4 interacting protein,
soluble
Munc18 Syntaxin-binding proteins, soluble,
three isoforms
Rabs Low-molecular-weight GTPases
involved in vesicle traffic, many
isoforms
Hrs-2 Soluble protein predicted to be
involved in vesicle traffic
SNAK Serine/threonine kinase showing
some substrate specificity for
SNAP-23
NSF, N-ethylmaleimide (NEM)-sensitive factor; SNAP, soluble
NSF attachment protein; t-SNARE, target soluble NSF attachment
protein receptor (SNARE); VAMP, vesicle-associated membrane pro-
tein; v-SNARE, vesicle SNARE; VAP-33, 33-kDa VAMP-associated
protein; SNAK, SNAP-23 kinase.
dues 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).
It was originally proposed that the SNARE proteins
would form a link between vesicle and target mem-
branes 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 mem-
branes (121). This has led to the suggestion that cis-
complexes of v- and t-SNARE may occur within the
same membrane, preventing the individual compo-
nents from engaging in trans-interactions with the
opposite membrane. The action of NSF and ␣SNAP is
to dissociate the cis-complexes using the energy re-
leased by ATP hydrolysis (Fig. 2) (7, 21). The final
INVITED REVIEW
fusion step depends on SNARE protein integrity but
appears to be independent of ATP hydrolysis, suggest-
ing that NSF is not involved at this stage (7, 21). A
Key
Reference
structure-function model of fusion has been proposed
whereby SNAREs in the docked conformation “zip up”
12 (Fig. 2) to form a tight, stable SNARE complex (40).
20
The complex involves a four-helix coiled-coil bundle
now described at atomic resolution (98). The free en-
10 ergy released by the formation of this exceptionally
stable complex is thought to be the source of the energy
86 used to fuse the two lipid bilayers (40). The elucidation
of the crystal structure of the SNARE complex led to an
108 alternative classification of SNAREs into Q- or
R-SNAREs, based on the presence of either a glu-
94 tamine (Q) or arginine (R) residue in the center of the
SNARE complex (29).
38
72
Downloaded from ajpcell.physiology.org on October 29, 2010
102
107
9
14
Fig. 1. Common domain structure of soluble N-ethylmaleimide-sen-
sitive factor attachment protein receptor (SNARE) proteins. A: syn-
taxins typically contain 3 coiled-coil domains. The most COOH ter-
minal (juxtamembrane) of these domains contains the conserved
glutamine (Q) residue and participates in the SNARE complex. Both
vesicle-associated membrane proteins (VAMPs) and syntaxins have
an extreme COOH-terminal transmembrane domain that protrudes
through the membrane, leaving the COOH terminus extracellular
(luminal). Similar to syntaxins, VAMPs also contain a juxtamem-
brane coiled-coil domain that participates in the SNARE complex,
but VAMP coiled-coil domains typically contain a conserved arginine
(R) residue. SNAP-25 and its homologs contain 2 coiled-coil domains,
1 at either end of the molecule, and both domains contain conserved
glutamine residues. In addition, these molecules have multiple cys-
teine residues (cccc) in the middle of the molecule that can be
palmitoylated to enhance the interaction of SNAP-25 with mem-
branes. B: the 3 SNARE proteins condense into a complex containing
4 ␣-helices, 2 contributed by SNAP-25 and 1 each by VAMP and
syntaxin. The helices align in a parallel fashion, presumably also
parallel to the plane of the membranes. The implication is that the
flexible linker between the 2 coiled-coil domains of SNAP-25 must
loop back around the complex to allow both domains to align in a
parallel fashion. N, NH2 terminus; C, COOH terminus.
 INVITED REVIEW
Fig. 2. Hypothetical steps in vesicle docking and fusion applied to
the glucose transporter 4 (GLUT-4) system. Preformed cis-SNARE
complexes on the plasmalemmal and vesicle membranes must first
be 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 the
plasma membrane (tethering) through as yet unknown molecules.
Rab4 may participate at this point, but tethering does not involve
SNARE proteins. Once tethered, the SNAREs can trans-associate,
causing the vesicle to become more tightly associated with the
plasma membrane (docking). Docking then leads to formation of the
classic SNARE complex on the way to fusion of the vesicle with the
plasma membrane.
Because SNARE proteins were first identified in
neuronal or neuroendocrine tissues, most of our infor-
mation 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 conser-
vation 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).
C879
GLUT-4 COMPARTMENTS AND THEIR SNARE
ISOFORMS
An important biological phenomenon involving ves-
icle-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 insu-
lin 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 com-
Downloaded from ajpcell.physiology.org on October 29, 2010
partments 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 en-
dosome, 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 addi-
tional mobilization of the specialized vesicular pool in
response to exercise. This diversity of fusion events
begs the question of whether similar or different mol-
ecules participate in GLUT-4 vesicle fusion with the
plasma membrane in each case. In the search for an-
swers 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
C880 INVITED REVIEW
(105). Another difference between neuronal/neuroen-
docrine 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).
THE PREFUSION STEPS
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 autoanti-
gen 1 (EEA1) protein may act as the tethering protein
engaged in binding endocytic vesicles to the early en-
dosome (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 num-
bers 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 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-L1
adipocytes, 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 cell
permeabilization (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 in
fat 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) 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 clos-
tridium 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 adipo-
cytes (17, 65, 68, 75) (Table 2).
Downloaded from ajpcell.physiology.org on October 29, 2010
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 com-
partment, 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 mi-
croinjected 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. Micro-
injection (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).
 INVITED REVIEW C881

Table 2. Effects of interfering with SNARE proteins on insulin-stimulated glucose uptake

and/or GLUT-4 translocation
GLUT-4 Glucose
Cell Type Reagent and Method of Introduction Translocation Uptake References

VAMP-2
3T3-L1 Botulinum toxin D by streptolysin O toxin Decreased n.d. 17
3T3-L1 Botulinum toxin B by streptolysin O toxin Decreased Decreased 99
3T3-L1 Cytoplasmic domain by streptolysin O toxin Decreased n.d. 17, 68
3T3-L1 NH2-terminal peptide by microinjection Decreased n.d. 65
3T3-L1 Tetanus toxin by microinjection Decreased n.d. 65
3T3-L1 Botulinum toxin B by microinjection Decreased n.d. 31, 65
3T3-L1 Botulinum toxin B by toxin-mediated uptake n.d. Decreased 18
3T3-L1 Cytoplasmic domain by adenoviral transfection Decreased n.d. 75
Rat adipocytes Tetanus toxin by electroporation No effect No effect 39
3T3-L1 NH2-terminal peptide by streptolysin O toxin Decreased n.d. 68
L6 myoblasts Tetanus toxin by transfection Decreased n.d. 85
Syntaxin 4
3T3-L1 Cytoplasmic domain by streptolysin O toxin Decreased n.d. 17
3T3-L1 Antibody by streptolysin O toxin n.d. Decreased 119
3T3-L1 Internal peptide (106–22) by microinjection Decreased n.d. 65

Downloaded from ajpcell.physiology.org on October 29, 2010

3T3-L1 Cytoplasmic domain by adenoviral transfection Decreased n.d. 75
3T3-L1 Cytoplasmic domain by microinjection Decreased n.d. 75
3T3-L1 Antibody by microinjection Decreased n.d. 101
SNAP-23
3T3-L1 Botulinum toxin E by toxin-mediated uptake n.d. No effect 18
3T3-L1 Botulinum toxin E by microinjection No effect n.d. 66
3T3-L1 COOH-terminal peptide by microinjection Decreased n.d. 87
3T3-L1 NH2-terminal antibody by microinjection Decreased n.d. 87
3T3-L1 COOH-terminal antibody by microinjection Decreased n.d. 34
3T3-L1 Full-length protein by microinjection Increased n.d. 34
3T3-L1 Full-length protein by streptolysin O toxin n.d. Increased 34
3T3-L1 Botulinum toxin E by microinjection Decreased n.d. 31
3T3-L1 Full-length protein by adenoviral transfection No effect No effect 58
3T3-L1 NH2-terminal 202 amino acids by adenoviral transfection Decreased Decreased 58
3T3-L1 NH2-terminal 161 amino acids by adenoviral transfection No effect No effect 58
GLUT-4, glucose transporter 4; n.d., not determined.

SNAP-23 function. The microinjected full-length protein en-

SNAP-23 shares both sequence and structural ho-
mology 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 transfec-
tion. 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 re-
ducing GLUT-4 translocation (31). In other studies, the
toxin was ineffective toward SNAP-23 (18, 66). Be-
cause 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
hanced 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 syn-
taxin 4 (1.15 ⫻ 106 copies of SNAP-23 per cell com-
pared 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. Exoge-
nous SNAP-23 may enhance the rate of fusion of
GLUT-4 vesicles with the plasma membrane by en-
hancing the formation of productive complexes with
syntaxin 4 and VAMP-2 (Table 2).
NSF and ␣SNAP
Unlike the expanded nature of the SNARE protein
families, NSF and ␣SNAP have very few apparent
homologs. High-resolution X-ray crystal structures
suggest that NSF may engage ␣SNAP as a lever to pry
the SNARE complex apart (129) (Table 1). This would
then allow SNAREs to form complexes between oppos-
ing membranes that are competent for fusion. Indeed,
NSF and ␣SNAP are found in rat adipocytes, and
C882
epitope-tagged versions of these proteins have been
used to immunoprecipitate SNARE complexes from
these cells. Such complexes contained syntaxin 4,
VAMP-2, VAMP-3, and SNAP-23 (105). Transfection of
a dominant negative mutant of NSF into rat adipocytes
resulted in plasma membrane levels of GLUT-4 after
insulin treatment that were not significantly different
from basal, nontransfected levels (45). However, the
level of plasma membrane GLUT-4 in basal cells ex-
pressing the dominant negative NSF was also lowered
significantly (45).
DO SNARES SUFFICE TO CAUSE GLUT-4 VESICLE
FUSION?
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, in-
terfering with VAMP-2, syntaxin 4, or SNAP-23 only
partly inhibited insulin-stimulated GLUT-4 transloca-
tion, 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 observa-
tion 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-ter-
minal 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 ac-
counted for by VAMP-2. Similarly, it is conceivable
that 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 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
Table 3. Reported SNARE complexes containing SNAP-23, VAMP-2, and syntaxin 4
System
Immunopurified from rat adipocyte cell lysates
Recombinant cytosolic domains, affinity purified
Recombinant cytosolic domains, size-exclusion purified,
SDS-PAGE
Recombinant cytosolic domains, affinity purified
Immunopurified from rat adipocyte cell lysates
Immunopurified from 3T3-L1 and COS cell lysates
Neuronal complex of syntaxin 1, VAMP-2, and SNAP-25
CD, circular dichroism; SPR, surface plasmon resonance.
INVITED REVIEW
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 micro-
calorimetry 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 ad-
dressed experimentally, yielding somewhat surprising
Downloaded from ajpcell.physiology.org on October 29, 2010
results (Table 3). Although there is general agreement
that a complex does form comprising SNAP-23, syn-
taxin 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 differ-
ences depend on the experimental design including,
importantly, whether the proteins used are recombi-
nant forms produced in bacteria or endogenous pro-
teins 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 com-
plex containing VAMP-2, SNAP-23, and syntaxin 4 in
0.5% SDS was detected using surface plasmon reso-
nance (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 overex-
pressed in COS cells (58).
SDS
Detection Method Cooperativity Resistance References
SDS-PAGE n.d. n.d. 105
SDS-PAGE None No 35
SDS-PAGE, CD n.d. No 128
SPR n.d. Yes 87
SDS-PAGE Enhanced by ␣SNAP n.d. 96
SDS-PAGE Yes, in COS cells n.d. 58
SDS-PAGE, CD, SPR Enhanced by SNAP-25 Yes 51
 INVITED REVIEW
HORMONAL REGULATION OF GLUT-4 VESICLE
INCORPORATION INTO TARGET MEMBRANES
An important question is whether any of the steps
involved in GLUT-4 vesicle incorporation into the
plasma membrane is regulated by insulin-derived sig-
nals. The occupied insulin receptor undergoes auto-
phosphorylation and then phosphorylates insulin-re-
ceptor substrate(s) (27, 69). This phosphorylation leads
to activation of phosphatidylinositol 3⬘-kinase to pro-
duce phosphatidylinositol 3,4-bisphosphate and phos-
phatidylinositol 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 physio-
logical conditions may halt GLUT-4 vesicles at the
docking state and prevent fusion. Isoproterenol pre-
treatment 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 main-
tained GLUT-4 in an occluded state, inaccessible to the
cell surface. Because the photolabel used has a prefer-
ential reactivity with active transporters, it was also
possible that the transporters were inactive but
present at the outer surface of the membrane. There-
fore, 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 isoproter-
enol pretreatment rendered the transporter inaccessi-
ble 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 re-
duced 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 (Ha-
yashi M, Bilan P, and Klip A, unpublished observa-
tions). Experiments are currently underway to deter-
mine 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 differ-
entiate between docking and fusion (48). GLUT-4 ves-
C883
icles 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 cal-
cium and could be prevented by including the recom-
binant soluble domain of syntaxin 4 in the binding
assay. Notably, plasma membranes derived from insu-
lin-stimulated cells were more effective than control
membranes in binding intracellular GLUT-4 myc (48).
Although this assay does not distinguish between dock-
ing and fusion, it may prove helpful in testing the
individual participation of enzymes known to be acti-
vated by insulin on the interaction of donor and accep-
tor membranes.
Phosphorylation
A plausible mechanism whereby insulin- or isoprot-
erenol-dependent signals could regulate the fusion ma-
chinery is through phosphorylation of its integral com-
Downloaded from ajpcell.physiology.org on October 29, 2010
ponents. Indeed, considerable efforts have been
dispensed toward defining which SNAREs are suscep-
tible to phosphorylation and by which kinases (Table
4). We have reported that syntaxin 4 is susceptible to
phosphorylation by the serine/threonine kinases pro-
tein kinase A, casein kinase II, and conventional pro-
tein kinase C in vitro (35). SNAP-23 is also phosphor-
ylated by conventional protein kinase C’s, but the
phosphorylation is inefficient (35). In addition, the
newly identified SNAP-23 kinase (SNAK) can phos-
phorylate SNAP-23 and syntaxin 4 (14).
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 selec-
tively 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 phosphoryla-
tion, 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) (Ta-
ble 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 fu-
sion. A cohort of proteins have been described to inter-
act with SNAREs, as described below and listed in
Tables 1 and 5.
Munc18c. The Munc18 proteins are mammalian ho-
mologs 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
C884 INVITED REVIEW

Table 4. Phosphorylation of SNAREs and ancillary proteins

Hormone Level/ Kinase Effect of
Protein System Regulation Stoichiometry Responsible Phosphorylation Reference

Syntaxin 4 Immune complex from None with INS Low Unknown Unknown ⴱ
3T3-L1 adipocytes or IPT
Syntaxin 4 Immune complex from n.d. Low Unknown, not Unknown 14
HeLa cells SNAK
Syntaxin 4 Recombinant protein/ n.a. 3.9 mol/mol Protein Inhibits binding to 35
purified kinase kinase A SNAP-23
Syntaxin 4 Recombinant protein/ n.a. 0.81 mol/mol Casein kinase Unknown 35
recombinant kinase II
Syntaxin 4 Recombinant protein/ n.a. ⬍0.05 mol/mol Protein Unknown 35
purified kinase kinase C
Syntaxin 4 Recombinant protein/ n.a. Low SNAK Unknown 14
recombinant kinase
SNAP-23 Recombinant protein/ n.a. ⬍0.01 mol/mol Protein Unknown 35
purified kinase kinase C
SNAP-23 Immune complex from None with INS Low Unknown Unknown ⴱ
3T3-L1 adipocytes or IPT
SNAP-23 Recombinant protein/ n.a. High SNAK Enhances binding to 14
recombinant kinase syntaxin 4
VAMP-2 Immune complex from None with INS Low Unknown Unknown ⴱ

Downloaded from ajpcell.physiology.org on October 29, 2010

3T3-L1 adipocytes
Pantophysin Immune complex from None with INS High Unknown Unknown 13
3T3-L1 adipocytes
77-kDa Bound to pantophysin
unknown immune complex INS increases Unknown Unknown Unknown 13
Ins, insulin; Iso, isoproterenol; n.a., not applicable. * Foster L and Klip A, unpublished observations.

clone similar genes from a 3T3-L1 cDNA library. One binding of syntaxin 4 to VAMP-2 (101, 103) and
of the proteins cloned by this method was Munc18c, SNAP-23 (3). Insulin causes the dissociation of a
which interacts specifically with syntaxins 2 and 4 but Munc18c/syntaxin 4 complex (103). A prediction of this
not syntaxins 1 or 3 (100, 102). Munc18c inhibits the observation is that once insulin causes the dissociation,

Table 5. Effects of interfering with SNARE-binding proteins on insulin-stimulated glucose uptake

or GLUT-4 translocation
GLUT-4 Glucose
Cell Type Reagent and Method Translocation Uptake Reference

Munc18c
3T3-L1 Full-length protein by adenoviral transfection Decreased n.d. 103
3T3-L1 Full-length protein by adenoviral transfection Decreased Decreased 100
3T3-L1 Syntaxin 4-binding peptide by microinjection Decreased n.d. 104
Synip
3T3-L1 COOH-terminal half by adenoviral Decreased Decreased 72
transfection/microinjection
3T3-L1 Full-length protein by adenoviral transfection/microinjection No effect No effect 72
3T3-L1 NH2-terminus half by adenoviral transfection/microinjection No effect No effect 72
Rab4
Rat adipocytes Peptide (191–210) by electroporation Decreased Decreased 93
Rat adipocytes Antibody by electroporation Decreased Decreased 93
Rat adipocytes Full-length protein by transfection Decreased n.d. 22
Rat adipocytes Membrane-association mutant by transfection Decreased n.d. 22
3T3-L1 Full-length protein by microinjection No effect n.d. 120
3T3-L1 GTP-binding mutant by microinjection Decreased n.d. 120
3T3-L1 GTPase mutant by microinjection No effect n.d. 120
3T3-L1 Antibody by microinjection Decreased n.d. 120
VAP-33
3T3-L1 Antibody by microinjection Decreased n.d. 33
L6 myoblasts Full-length protein by transfection Decreased n.d. 33
Pantophysin
3T3-L1 Antibody by microinjection Decreased n.d. ⴱ
* Foster LJ, Cheatham B, and Klip A, unpublished observations.
 INVITED REVIEW C885

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 intro-
duced into 3T3-L1 adipocytes by adenoviral transfec-
tion 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 pro-
tein-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 sug-
gest that the displaced, endogenous Munc18c catalyzes
fusion (104) (Fig. 3).
Synip. The recently cloned synip is a syntaxin 4-in-
teracting 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
phorylation 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 insu-
lin-sensitive tissues or whether SNAK is activated by
insulin. Results of in vivo phosphorylation do not sup-
port 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 inhib-
its 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

Downloaded from ajpcell.physiology.org on October 29, 2010

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 con-
ferred by the NH2-terminal half of synip, whereas the
COOH-terminal half modulates GLUT-4 translocation
(72). Despite having unrelated primary sequences, sy-
nip and Munc18c regulate the availability of syntaxin 4
for 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 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 phos-
from 3T3-L1 cells and, similarly to synaptophysin,
binds VAMP-2 (13). Interestingly, although pantophy-
sin itself was not phosphorylated, a 77-kDa phospho-
protein associates with pantophysin upon treatment of
cells with insulin (13). This result suggests a potential
regulation of pantophysin by insulin. Preliminary re-
sults from our laboratory suggest that pantophysin
availability is required for GLUT-4 vesicle fusion (Fos-
ter 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 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 pro-
tein with the bulk of the molecule in the cytosol. Two
isoforms 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 and
synip 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 from
the vesicles, Rab4 organizes the proteins on the vesicles into conformations required for fusion. A 33-kDa
VAMP-2-associating protein (VAP-33) may also be involved as a chaperone for VAMP-2. Insulin causes the
formation of actin ruffling and the dissociation of Munc18c and synip from syntaxin 4. Once brought into the
proximity of fusion machinery at the plasma membrane by the cytoskeleton, the GLUT-4 vesicle can dock and
subsequently fuse, exposing GLUT-4 to the extracellular milieu.
C886 INVITED REVIEW
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. Interest-
ingly, 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 adi-
pocytes 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 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 by
overexpression of specific t-SNAREs, and the Rab
Ypt1p is required for v-SNARE-t-SNARE complex for-
mation. 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 endomem-
branes (22, 36). Introduction of wild type or mutants of
Rab4 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 docking
and the actin-based cytoskeleton has been established.
Rabphilin, a Rab3 effector, interacts with the actin-
bundling protein ␣-actinin (57), and Rab8 promotes
polarized membrane transport through reorganization
of actin filaments (79).
The Cytoskeleton
The actin cytoskeleton has been repeatedly impli-
cated 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 secre-
tory 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 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 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 mol-
ecules (59). Preventing cortical actin structure forma-
tion through transient expression of a dominant nega-
tive Rac mutant abrogated externalization of GLUT-4
(59). In nontransfected muscle cells, GLUT-4 is in-
serted 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 obser-
vations). In adipocytes, a requirement for an organized
cytoskeleton in GLUT-4 traffic has also been demon-
strated (76). It will be interesting to determine
whether GLUT-4 vesicles, once delivered by the cy-
toskeleton to the vicinity of the plasma membrane,
Downloaded from ajpcell.physiology.org on October 29, 2010
require Rab4 as the tethering molecule leading to
SNARE complex formation.
CONCLUSION
GLUT-4 vesicle fusion bears similarities to and dif-
ferences 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, syn-
taxin 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 indepen-
dent 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 phosphor-
ylation. However, it is conceivable that subtle regula-
tion may still occur by this means. In contrast, there is
emerging evidence that ancillary proteins such as
Munc18c, synip, VAP-33, and pantophysin may regu-
late the availability of VAMP-2 or syntaxin 4 for pro-
ductive GLUT-4 fusion. Cytoskeletal tethering of vesi-
cles 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 ves-
icle 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 resis-
tance. Both reports suggest that these increases might
be adaptive changes attempting to overcome the de-
fects in GLUT-4 traffic that underlie insulin resistance.
We acknowledge the Juvenile Diabetes Foundation and the Med-
ical Research Council of Canada for funding.
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. Bio-
chem Biophys Res Commun 215: 321–328, 1995.
 INVITED REVIEW
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.
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.
4. Armstrong J. How do Rab proteins function in membrane
traffic. Int J Biochem Cell Biol 32: 303–307, 2000.
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.
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-phospho-
inositide-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-de-
pendent 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.
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.
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.
11. Bernstein BW, DeWit M, and Bamburg JR. Actin disassem-
bles reversibly during electrically induced recycling of synaptic
vesicles in cultured neurons. Mol Brain Res 53: 236–250, 1998.
12. Block MR, Glick BS, Wilcox CA, Wieland FT, and Roth-
man JE. Purification of an N-ethylmaleimide-sensitive protein
catalyzing vesicular transport. Proc Natl Acad Sci USA 85:
7852–7856, 1988.
13. Brooks CC, Scherer PE, Cleveland K, Whittemore JL,
Lodish HF, and Cheatham B. Pantophysin is a phosphopro-
tein 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. Phosphor-
ylation 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 glu-
cose 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.
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.
C887
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.
21. Colombo MI, Taddese M, Whiteheart SW, and Stahl PD. A
possible predocking attachment site for N-ethylmaleimide-sen-
sitive 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 Ob-
berghen 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.
23. Cormont M, Tanti JF, Zahraoui A, van Obberghen E,
Tavitian A, and Le Marchand-Brustel Y. Insulin and oka-
daic 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 mecha-
nisms involved in GLUT4 translocation in muscle during insu-
lin and contraction stimulation. Adv Exp Med Biol 441: 63–71,
1998.
25. Douen AG, Ramlal T, Rastogi S, Bilan PJ, Cartee GD,
Vranic M, Holloszy JO, and Klip A. Exercise induces recruit-
ment of the “insulin-responsive glucose transporter”. Evidence
for distinct intracellular insulin- and exercise-recruitable
Downloaded from ajpcell.physiology.org on October 29, 2010
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-con-
taining intracellular vesicles. Exp Cell Res 253: 55–62, 1999.
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. Con-
served 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 insu-
lin-stimulated rat adipose cells: evidence that heterotrimeric
GTP-binding proteins regulate the fusion of docked GLUT4-
containing vesicles. Biochem J 343: 571–577, 1999.
31. Foran PG, Fletcher LM, Oatey PB, Mohammed N, Dolly
JO, and Tavare JM. Protein kinase B stimulates the translo-
cation of GLUT4 but not GLUT1 or transferrin receptors in
3T3-L1 adipocytes by a pathway involving SNAP-23, synapto-
brevin-2, and/or cellubrevin. J Biol Chem 274: 28087–28095,
1999.
32. Foster LJ, Khayat ZA, and Klip A. Insulin-dependent intra-
cellular 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.
34. Foster LJ, Yaworsky K, Trimble WS, and Klip A. SNAP23
promotes insulin-dependent glucose uptake in 3T3-L1 adipo-
cytes: 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 syn-
taxin-4, SNAP23, and VAMP-2 and their regulation by phos-
phorylation. Biochemistry 37: 11089–11096, 1998.
36. Goalstone ML, Leitner JW, Golovchenko I, Stjernholm
MR, Cormont M, Le Marchand-Brustel Y, and Draznin B.
Insulin promotes phosphorylation and activation of gera-
nylgeranyltransferase 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 mam-
C888 INVITED REVIEW
malian 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.
39. Hajduch E, Aledo JC, Watts C, and Hundal HS. Proteolytic
cleavage of cellubrevin and vesicle-associated membrane pro-
tein (VAMP) by tetanus toxin does not impair insulin-stimu-
lated glucose transport or GLUT4 translocation in rat adipo-
cytes. Biochem J 321: 233–238, 1997.
40. Hanson PI, Heuser JE, and Jahn R. Neurotransmitter re-
lease - four years of SNARE complexes. Curr Opin Neurobiol 7:
310–315, 1997.
41. Hashiramoto M and James DE. Characterization of insulin-
responsive GLUT4 storage vesicles isolated from 3T3-L1 adipo-
cytes. Mol Cell Biol 20: 416–427, 2000.
42. Haworth RA, Hunter DR, and Berkoff HA. Insulin stimu-
lates deoxyglucose transport in adult rat heart cells in the
absence of Ca2⫹. FEBS Lett 141: 37–40, 1982.
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.
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 ATP-
ase-deficient mutant of N-ethylmaleimide sensitive fusion pro-
tein (NSF) in rat adipose cells impairs insulin-stimulated
GLUT4 translocation. Diabetes 47: A241, 1998.
46. Hirshman MF, Goodyear LJ, Wardzala LJ, Horton ED,
and Horton ES. Identification of an intracellular pool of glu-
cose transporters from basal and insulin-stimulated rat skele-
tal 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.
48. Inoue G, Cheatham B, and Kahn CR. Development of an in
vitro reconstitution assay for glucose transporter 4 transloca-
tion. 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-asso-
ciated protein 25) A and B isoforms in addition to syntaxin 4
and synaptobrevins 1 and 2. Biochem J 317: 945–954, 1996.
50. Jahn R, Hanson PI, Otto H, and Ahnert-Hilger G. Botuli-
num and tetanus neurotoxins: emerging tools for the study of
membrane fusion. Cold Spring Harb Symp Quant Biol 60:
329–335, 1995.
51. Jahn R and Südhof TC. Membrane fusion and exocytosis.
Annu Rev Biochem 68: 863–911, 1999.
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.
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 synap-
tobrevin 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.
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.
57. Kato M, Sasaki T, Ohya T, Nakanishi H, Nishioka H,
Imamura M, and Takai Y. Physical and functional interac-
tion 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 transloca-
tion 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.
61. Klip A and Ramlal T. Cytoplasmic Ca2⫹ during differentia-
tion 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.
63. Lemieux K, Han XX, Dombrowski L, Bonen A, and Ma-
rette A. The transferrin receptor defines two distinct contrac-
tion-responsive GLUT4 vesicle populations in skeletal muscle.
Diabetes 49: 183–189, 2000.
Downloaded from ajpcell.physiology.org on October 29, 2010
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.
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 trans-
location in 3T3-L1 adipocytes. Biochem Biophys Res Commun
237: 388–393, 1997.
67. Maier VH, Melvin DR, Lister CA, Chapman H, Gould GW,
and Murphy GJ. v- and t-SNARE protein expression in mod-
els of insulin resistance. Diabetes 49: 618–625, 2000.
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.
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.
71. Mills IG, Jones AT, and Clague MJ. Regulation of endosome
fusion. Mol Membr Biol 16: 73–79, 1999.
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.
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. Molec-
ular cloning and characterization of mammalian homologues of
vesicle-associated membrane protein-associated (VAMP-associ-
ated) proteins. Biochem Biophys Res Commun 254: 21–26,
1999.
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 translo-
cation in adipocytes. Mol Cell Biol 17: 2425–2435, 1997.
 INVITED REVIEW
76. Omata W, Shibata H, Li L, Takata K, and Kojima I. Actin
filaments play a critical role in insulin-induced exocytotic re-
cruitment but not in endocytosis of GLUT4 in isolated rat
adipocytes. Biochem J 346: 321–328, 2000.
77. Oyler GA, Higgins GA, Hart RA, Battenberg E, Billings-
ley 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.
78. Patki V, Virbasius J, Lane WS, Toh BH, Shpetner HS, and
Corvera S. Identification of an early endosomal protein regu-
lated 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 reorga-
nization of actin and microtubules in fibroblasts. J Cell Biol
135: 153–167, 1996.
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 re-
cruited by insulin and muscle contractions. J Cell Biol 142:
1429–1446, 1998.
81. Quinones B, Riento K, Olkkonen VM, Hardy S, and Ben-
nett MK. Syntaxin 2 splice variants exhibit differential expres-
sion patterns, biochemical properties and subcellular localiza-
tions. 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-medi-
ated translocation of glucose transporters from intracellular
membranes to plasma membranes: sole mechanism of stimula-
tion of glucose transport in L6 muscle cells. Biochem Biophys
Res Commun 157: 1329–1335, 1988.
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.
89. Scales SJ, Chen YA, You BY, Patel SM, Doung YC, and
Scheller RH. SNAREs contribute to the specificity of mem-
brane fusion. Neuron 26: 457–464, 2000.
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-sensi-
tive 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 car-
boxyl-terminal domain inhibits insulin action on glucose trans-
port 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 neurotrans-
mitter release. Science 269: 1580–1583, 1995.
C889
95. Söllner T, Whiteheart SW, Brunner M, Erdjument-Bro-
mage H, Geromanos S, Tempst P, and Rothman JE. SNAP
receptors implicated in vesicle targeting and fusion. Nature
362: 318–324, 1993.
96. St-Denis JF, Cabaniols JP, Cushman SW, and Roche PA.
SNAP-23 participates in SNARE complex assembly in rat adi-
pose cells. Biochem J 338: 709–715, 1999.
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.
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.
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-con-
taining vesicles inhibits the translocation of GLUT4 in 3T3-L1
adipocytes. Biochem Biophys Res Commun 220: 740–745, 1996.
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
Downloaded from ajpcell.physiology.org on October 29, 2010
CW, and James DE. Characterization of Munc-18c and syn-
taxin-4 in 3T3-L1 adipocytes. Putative role in insulin-depen-
dent movement of GLUT-4. J Biol Chem 272: 6179–6186, 1997.
102. Tellam JT, McIntosh S, and James DE. Molecular identifi-
cation of two novel Munc-18 isoforms expressed in non-neuro-
nal 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 pep-
tidase 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. Identi-
fication of SNAP receptors in rat adipose cell membrane frac-
tions and in SNARE complexes co-immunoprecipitated with
epitope-tagged N-ethylmaleimide-sensitive fusion protein. Bio-
chem J 320: 429–436, 1996.
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 oligonucle-
otide strategy: molecular cloning of YPT-related cDNAs from a
rat brain library. Proc Natl Acad Sci USA 84: 8210–8214, 1987.
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.
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.
111. Uphues I, Kolter T, Goud B, and Eckel J. Insulin-induced
translocation of the glucose transporter GLUT4 in cardiac mus-
cle: studies on the role of small-molecular-mass GTP-binding
proteins. Biochem J 301: 177–182, 1994.
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 sub-
apical actin cytoskeleton regulates secretion and membrane
retrieval in pancreatic acinar cells. J Cell Sci 112: 81–96, 1999.
C890 INVITED REVIEW
114. Vannucci SJ, Nishimura H, Satoh S, Cushman SW, Hol-
man GD, and Simpson IA. Cell surface accessibility of
GLUT4 glucose transporters in insulin-stimulated rat adipose
cells. Biochem J 288: 325–330, 1992.
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.
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 cellubre-
vin in rat skeletal muscle and in a muscle cell line. Biochem J
304: 139–145, 1994.
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.
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 pro-
tein Rab4 is involved in insulin-induced GLUT4 translocation
and actin filament rearrangement in 3T3-L1 cells. Endocrinol-
ogy 138: 4941–4949, 1997.
121. Walch-Solimena C, Blasi J, Edelmann L, Chapman ER,
Fischer von Mollard G, and Jahn R. The t-SNAREs syn-
taxin 1 and SNAP-25 are present on organelles that participate
in synaptic vesicle recycling. J Cell Biol 128: 637–645, 1995.
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.
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.
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 pro-
tein that binds to VAMP. Biochem J 333: 247–251, 1998.
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 Bio-
phys Res Commun 230: 64–68, 1997.
128. Yang B, Gonzalez L Jr, Prekeris R, Steegmaier M, Advani
Downloaded from ajpcell.physiology.org on October 29, 2010
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.
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.