THEJOURNAL

OF BIOLOGICAL
CHEMISTRY Vol. 267, No. 9,Issue of March 25, pp. 6278-6285,1!392
0 1992 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S. A.

Immunocytochemical and Biochemical Studies of GLUT4 in Rat

Skeletal Muscle*
(Received for publication, August 13, 1991)

Kenneth J. Rodnick$$, J a n W.Slotnll, Daniel R. Studelska**, DavidE.HanpeterS$$&

Linda J. Robinson**, Hans J. Geuzell, and DavidE. James**qll
From the $Departments of Medicine, **Cell Biologyand Physiology, and $$Surgery, Washington University School of Medicine,
St. Louis, Missouri 63110 and the YDepartment of Cell Biology, Medical School, University of Utrecht, 3584 CX,
Utrecht, The Netherlands

In muscle and adipocytes, glucose transport is regu- cellular compartment to thecell surface (1-13). In adipocytes,
lated by the translocation of insulin regulatableglucose subcellular fractionation studies aswell as immunocytochem-
transporters (GLUT4) between an intracellular com- ical observations have indicated that GLUT4 is essentially
partment and the cell surface.In these studies we haveabsent from the plasma membrane under basal conditions,
characterized the cellular compartments containing whereas 40% of the totalGLUT4 is present at thecell surface
GLUT4 in rat skeletal muscle. Immunocytochemical after insulin stimulation (3, 8). Immunocytochemical studies
studies showed that in unstimulated muscle, GLUT4 have also provided insights about the intracellular sorting of
was not present in surfacemembranes. Tubulo-vesic- GLUT4. In the unstimulatedstate, GLUT4 resides exclu-
ular structures clustered in the transGolgi reticulum sively in small tubulo-vesicular (TV)’ structures that are
were enriched inGLUT4. GLUT4 underwent translo- associated with the trans Golgi reticulum (TGR) and other

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cation to the sarcolemma response in to combined stim-
ulation with insulinand exercise. Using immunoisola- related organelles in the cell (8). With insulin stimulation,
tion, the intracellular GLUT4 vesicles (IRGTV) were these structures fuse with the plasma membrane, exposing
purified 300-fold over the cell homogenate. IRGTV GLUT4 to the cell surface. During continuous stimulation,
from unstimulated muscle were not enriched in mark- GLUT4 entersthe endocytic pathway via clathrin-coated
ers specific for the sarcolemma, transverse tubules, vesicles and may recycle back to thecell surface in a manner
sarcoplasmic reticulumor mitochondria; this wascon- similar to cell surface receptors (8).
firmed using gel filtration chromatography. Insulin This model of GLUT4 localization and movement appears
resulted ina 40% decrease in GLUT4 levelsin IRGTV to operate in white’ and brown adipocytes (8),as well as in
confirming that this represents the intracellularcom- cardiac myocytes (9). In skeletal muscle, however, the situa-
partment of GLUT4. GLUT4 is a major component of tion is not clear. A recent immunocytochemical study per-
the IRGTV, constituting at least 5% of total vesicle formed on unstimulated humanmuscle suggests that GLUT4
protein. A subset of polypeptides are also markedly resides in the triad region, and there was little evidence for
enriched in the muscle IRGTV. In conclusion, these GLUT4 translocation to the cell surface after insulin stimu-
data suggest that translocation of GLUT4 from intra- lation (14). Furthermore, subcellular fractionation studies in
cellular tubulo-vesicular structures is the majormech- stimulated skeletal muscle have demonstrated only a modest
anism by which insulin and exercise regulate muscle increase (2-3-fold) in GLUT4 at thecell surface, which cannot
glucose transport. account for the increase in glucose transport measured under
the same conditions (5-7). Due to this discrepancy, it has
been proposed that intrinsic activation of the transport ca-
Considerable evidence suggests that insulin stimulates glu- pacity of GLUT4 must occur with stimuli such as insulin and
cose transport in muscle and adipocytes by triggering the exercise.
translocation of glucose transporters (GLUT4) from an intra- It is important to determine if GLUT4 functions differently
in skeletal muscle compared to other cell types because skel-
* This research was supported by Grants DK-42503 and DK-18986 etal muscle makes the most significant contribution to insu-
from the National Institutes of Health and theWashington Univer- lin-stimulated glucose utilization in humans and the rat (15-
sity Diabetes Research and Training Center. Preliminary data from 17). It is possible that thediscrepancies described above may
this study were presented at the 14thInternational Diabetes Feder- reflect technical problems specific to skeletal muscle fraction-
ation Congress in conjunction with the 51st Annual Meeting of the ation. In thepresent study, we address the question of GLUT4
American Diabetes Association, June 23-28, 1991, Washington, D.C.
The costs of publication of this article were defrayed in part by the localization in skeletal muscle using two different approaches.
payment of page charges. This articlemust therefore be hereby First, we have employed immunocytochemical methods that
marked “advertisement” in accordance with 18 U.S.C. Section 1734 were used previously on insulin-responsive tissues (8, 9).
solely to indicate this fact. Second, we have developed specific fractionation procedures
J Supported by the National Institutes of Health, Institutional using immunoadsorption or gel filtration chromatography for
National Research Service Award AG-00078.
11 Supported by a grant from the Juvenile Diabetes Foundation.
JJ Supported by the National Institutes of Health, Institutional The abbreviations used are: TV, tubulo-vesicular; TGR, trans
National Research Service Award GM-07602-13. Golgi reticulum; HEPES, 4-(2-hydroxyethyl)-l-piperazineethanesul-
VI Recipient of a Career Development Award from the Juvenile fonic acid; BSA, bovine serum albumin; PBS, phosphate-buffered
Diabetes Foundation. To whom correspondence should be addressed saline; IRGTV, insulin regulatable glucose transporter vesicles;
Dept. of Cell Biology and Physiology, Washington University School GTPrS, guanosine 5’-O-(thiotriphosphate).
of Medicine. Box 8228,660 S. Euclid Ave., St. Louis, MO,63110. * Immunocytochemical localization of GLUT4 in white adipose is
Tel.: 314-362-6980;Fax: 314-362-7463. similar to thatin brown adipose tissue (Jan Slot, unpublished data).

6278
 GLUT4 in Muscle
the characterizationof intracellular GLUT4in skeletal mus-
cle. The dataobtained using bothapproachesshow that
GLUT4 distribution in skeletal muscle is similar to that in
adipocytes and cardiocytes.The improved fractionation meth-
ods also provide evidence that conventional techniques may
be inadequate for studying the distribution and translocation
of GLUT4 in skeletal muscle. Immunoadsorptionof intracel-
lular GLUT4 has allowedus to characterize the TV elements
in which GLUT4 resides, indicating that thiscompartment is
unique comparedto other organelles inskeletal muscle.
EXPERIMENTALPROCEDURES
Materials-Male Wistar rats weighing 150-175 g were purchased
from Sasco (Omaha, NE). 'T-Labeled goat anti-rabbit IgG and goat
anti-mouse IgG were obtained from ICN Radiochemicals (Irvine,
CA). Affinity purified goat anti-mouse or goat anti-rabbit IgG were
obtained from East Acres Biologicals (Southbridge, MA). Magnetic
beads coated with sheep anti-mouse IgG and magnetic particle con-
centrators were obtained from Dynal (Great Neck, NY). Reagents for
sodium dodecyl sulfate-polyacrylamide gel electrophoresis were ob-
tained from Bio-Rad. Crude collagenase from Clostridium histolyti-
cum (lot no. 475057) wasobtained from Worthington (Freehold, NJ).
Purified porcine insulin was obtained from Eli Lilly (Indianapolis,
IN). All other chemicals were obtained from Sigma.
Immunocytochemistry-Immunocytochemistry was performed on
soleus muscle isolated from control or stimulated rats. Stimulation
consisted of an intraperitoneal injection of insulin (8 units/kg) and
D-glucose (1g/kg) 30 min prior to tissue removal as well as treadmill
exercise (24 m/min) during the last 25 min prior to tissue removal.
Control animals, which were fasted overnight, received an injection
of saline. Rats were then anesthetized (Nembutal, 90 mg/kg body
weight) and subjected to whole body fixation with 2% paraformal-
dehyde, 0.2% glutaraldehyde in 0.1 M sodium phosphate buffer, pH
7.4 as previously described (8).The soleus muscle was dissected out
and ultrathin cryosections prepared (8), which were labeled with a
GLUT4 antibody (18) followedby swine anti-rabbit IgG (Nordic,
Tilburg, The Netherlands) and then protein A-gold. In some cases
this was followed by labelling for rat albumin using protein A-gold of
different size. The albumin labeling was used to mark the endocytic
pathway as well as theextracellular space.
Tissue Preparation for Biochemical Studies-After an overnight
fast, animals were anesthetized with sodium pentobarbital (50 mg/kg
body weight, intraperitoneal injection). The skin was removed and
the gastrocnemius and plantaris muscles were rapidly frozen in situ
using an aluminum clamp cooled to the temperature of liquid N,.
Frozen muscles were weighed and pulverized at the temperature of
liquid N,. The muscle powder was homogenized in ice-cold buffer (5
ml/g muscle) containing 20 mM HEPES, 1 mM EDTA, and 250 mM
sucrose (HES), pH 7.4, using a two-step process. The tissue was
initially homogenized using a Tekmar Tissuemizer (2 X 10 s) at 75%
of maximum output and thensubjected to 10 passes of a tight fitting
Potter-Elvehjem homogenizer at 800 revolutions/min. The homoge-
nate was centrifuged for 15 min at 3,000 X g in a Sorvall SS-34 rotor
a t 4 "C. The resultant pellet from this centrifugation was resuspended
in 10 volumes of HES buffer, rehomogenized using the Potter-El-
vehjem homogenizer, and centrifuged at 3,000 X g for 15 min as above.
Two additional extractions of the 3,000 X g pellet were performed,
and all the supernatants were combined and centrifuged a t 184,000
X g in a Beckman 50.2 Ti rotor for 90 min. The resulting high speed
pellet was resuspended in HES buffer to a final concentrationof 5-8
mg/ml and kept at 4 'C. This membrane fraction was used as the
starting material for subsequent immunoisolation and gel filtration
of intracellular GLUT4-containing vesicles. Protein concentrations
were determined using the bicinchoninic acid reagent (Pierce Chem-
ical Co.) or colloidal gold (Diversified Biotech, Newton Centre, MA)
according to manufacturer's instructions.
Antibodies-Both monoclonal (1F8) and polyclonal (R820) anti-
bodies specific for GLUT4 (3, 12) were employed in these studies.
Briefly, monoclonal antibody 1F8 was produced after immunization
of mice with partially purified intracellular glucose transporter-con-
taining vesicles from adipocytes (3). Anti-GLUT4 (R820), and anti-
GLUT1 (R493) sera from rabbits were generated against peptides
corresponding to the final 12 amino acids of the respective carboxyl
termini (12, 18). The anti-GLUT1 antibody was affinity purified
using the peptide antigen coupled to acrylamide beads (12). A rabbit
6279
polyclonal antibody generated against a synthetic peptide for the a
subunit of Na,K-ATPase was obtained from Dr. Robert Mercer at
the Washington University School of Medicine, St. Louis, MO. A
rabbit polyclonal antibody specific for 5'-nucleotidase was obtained
from Dr. Paul Luzio, University of Cambridge, Cambridge, United
Kingdom. A monoclonal antibody against the Ca2+-ATPaseprotein
was obtained from Dr. Kevin Campbell at the University ofIowa
College of Medicine, Iowa City, IA. Dr. Mario Rosenblatt from
Santiago, Chile provided a monoclonal antibody, tt28, whichwas
against a specific transverse tubule protein (19). A rabbit polyclonal
antibodyagainst succinate dehydrogenase was obtained fromDr.
John Lawrence, Washington University School of Medicine. Anti-
IGF I1 receptor antisera was obtained from Dr. Rob Baxter, Sydney
University, Sydney, Australia.
Zmmunoisolation of GLlJT4-containing Vesicles-Magnetic beads
(4.5-pm diameter) with sheep anti-mouse IgG bound to the surface
were used for immunoadsorption. In each of the steps described below,
beads were separated from the supernatantusing a magnetic particle
concentrator. Beads were blocked with BSA (10 mg/ml in PBS) for
30 min a t 22 "C, washed three times for 20 min each at 4 "C with
PBS, pH7.4. Ascites fluid (1F8) was added to thebeads (1 p1/4 X lo5
beads) and incubated for 14 h at 4 "C in PBS containing BSA (1mg/
ml), 1 mM EDTA, and 0.02% sodium azide using slow end over end
rotation (25 rpm). Beads were washed three times for 20 min each at
4 'C in PBS. Control beads were treated identically except that 1F8
was not added. Beads were then incubated with the 184,000 X g
muscle pellet suspension for 4 h a t 4 'C in PBS/BSA (1 mg/ml),
EDTA (1 mM), pH 7.4. This incubation was performed in a volume
of 1 ml containing membranes a t 100 pg/ml and 2 X lo7 beads. The
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beads were washed twice for 20 min at 4 "C with 0.5 ml of PBS. All
supernatants were saved and pelleted a t 200,000 X g for 30 min in a
Beckman TL-100 rotor. Both magnetic beads and pelleted mem-
branes were resuspended in PBS and prepared for sodium dodecyl
sulfate-polyacrylamide gel electrophoresis.
Gel Filtration-Sephacryl S-1000 was used to separate GLUT4-
containing vesicles from other muscle organelles. A column (1.6 X 65
cm) of Sephacryl S-1000 was equilibrated at 4 "C with a buffer
containing filtered (0.45 pm) 20 mM HEPES, 1 mM EDTA, 250 mM
sucrose, and 0.02% sodium azide, pH 7.4. The column was previously
conditioned with BSA and several aliquots of microsomes from rat
muscle and brain. Fractionation of intracellular vesicles containing
GLUT4 was performed by loading the column with 5 mg of protein
from the resuspended 184,000 X g pellet in a total volume of 3 ml.
The column was eluted a t 15 ml/h into 3-ml fractions. Column
fractions were used for protein determination and measurement of
various markers by immunoblotting. Following experimental runs,
the column was equilibrated with elution buffer containing 0.01 M
sodium dodecyl sulfate (20). Polystyrenemicrospheres of known size
(Polysciences, Inc., Warrington, PA) were then used to calibrate the
column. Eluted microspheres were detected by absorbance and scat-
tering a t 260 nm. A linearizing inverse error function plot (21) fitting
bead size and elution volume, yielded estimates of particle size. The
fluid volume of the column, required for constructing this plot, was
estimated from the elution position of tritiated water.
SDS-PAGE and Immunoblotting-Samples were immunoblotted
as previously described (3, 12). For GLUT1, GLUT4, and tt28deter-
minations, non-reducing 10% polyacrylamide gelswereused. For
Ca2+-ATPaseand Na,K-ATPase, samples were reduced with 20 mM
dithiothreitol, incubated at 65 "C for 20 min, and run on 7% gels.
Immunoblotting was performed using saturating concentrations of
each antibody. Autoradiographs were obtained by exposing nitrocel-
lulose sheets to preflashed Kodak X-OMAT AR-film and Cronex
Lightning Plus Enhancing screens at -70 "C. Autoradiographs were
quantitated by direct counting of excised bands or scanning laser
densitometry. Each autoradiograph was exposed for an appropriate
duration to ensure that scanning was performed over a linear range
for each protein.
Analysis of IRGTV Polypeptide Composition-To analyze the pro-
tein composition of the IRGTV, immunoisolation of the IRGTV was
performed using cellulose rather than magnetic beads as the solid
support. Cellulose was activated and goat anti-mouse or goat anti-
rabbit IgG bound by the method of Hales and Woodhead (22). Protein
A-agarose (Bio-Rad) column purified 1F8 was coupled to the goat
anti-mouse cellulose for immunoisolation of the IRGTV. Goat anti-
rabbit cellulose was utilized as a control. Cellulose was incubated
with the 184,000 X g muscle fraction for 3 h at 4 "C in PBS with 1
mg/ml fish gelatin. The cellulose wasseparated from the supernatant
by centrifugation at 12,000 X g for 3 min. The cellulose was washed
6280 GLUT4 in Muscle
twice with 1 ml of 1 M sodium chloride, followed by 1 ml of PBS, a t
4 "C. Non-reduced samples were run on 5-20% polyacrylamide gra-
dient gels, which were silver stained by the method of Oakley et al.
(23).
Electron Microscopy of Zmmunoisolnted Vesicles-Magnetic beads
containing GLUT4 vesicles and corresponding control beads were
initially diluted in PBS and then fixed in a solution of 0.1 M sodium
phosphate buffer, pH 7.4, containing 2% paraformaldehyde and 0.2%
glutaraldehyde for 30 min. The beads were placed on a thin slab of
fixed rabbit lung and quick frozen by impact against aliquid helium-
cooled copper block using a freezing machine (24). The sample was
freeze-fractured and deep etched for 4 min at -100 "C in a standard
Blazer's freeze etchunit and replicated with 2 nm of platinum
deposited at 24" above the horizontal. Replicas were cleared by
immersion in household bleach, rinsed in water, and mounted on
formvar-coated grids as described previously (25). Replicas were
viewed at 100 kV in a standard transmission electron microscope and
photographed a t ?lo" of tilt toobtain stereoimages. All images were
printed as photographic reversals (26). Vesicle sizing was performed
using photographic enlargements and a calibrated lox magnifier.

RESULTS
Immunocytochemistry-In control soleus muscle, GLUT4
labeling was pronounced in regions along the cell borders, in
agreement with previous studies using light microscopy (27).
Using electron micrographs, these regions were identified as

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clusters of tubulo-vesicular elements, often in close proximity
t o the Golgi cisternae, probably as partof the TGR (Fig. 1A).
Large vacuoles were often associated with the TV clusters
(Fig. 1, A and C). These vacuoles appear to be endosomes
because they are labeled with the anti-albumin antibody (Fig.
1C). Isolated GLUT4 positive vesicles werescattered through-
out the cytoplasm, often near the sarcolemma (Fig. lB), but
occasionally between the myofibrils near the transverse tu-
bules (Fig. 2). Very little GLUT4 labeling of the plasma
membrane was detected in basal cells (Fig. 1A). After stimu-
lation therewas a marked increase in GLUT4 labeling of the
sarcolemma (Fig. 1B) but not in the transverse tubules. No
significant GLUT4 labeling was associated with the sarco-
plasmic reticulum either inthe basal state (Fig. 2) or following
stimulation (data not shown).
Subcellular Fractionation-To substantiate the immuno-
cytochemical data (Figs. 1and 2), a procedure was developed
for partially purifying the intracellular IRGTV from skeletal
muscle. Because muscle glucose transport is acutely activated
by contractile activity (28), special precautions were observed
during muscle dissection in order to maintain unstimulated
conditions (see "Experimental Procedures"). Muscle homog-
enates were centrifuged at low speed (3000 X g) to remove
large aggregates of connective tissue and nuclei (see below).
The resulting supernatant was centrifuged at high speed to
yield a membrane fraction (184,000 x g pellet) that was 7-8-
fold enriched in GLUT4 and 2-3-fold enriched in Na,K-
ATPase, Ca2+-ATPase,tt28, and GLUT1 (Table I). The low
speed pellet contained <40% of total GLUT4 and >50% of
markers corresponding to the cell surface and sarcoplasmic
reticulum. In agreement with other studies in skeletal muscle
(29,30) repeated extraction could not liberate more than 20-
30% of surface membranes from this low speed pellet, presum-
ably because they remain attached to theextracellular matrix
or cytoskeletal and tubular networks. Using immunocyto-
chemistry (Figs. 1and 2) very little GLUT4 labeling of surface
membranes was detected under basal conditions. Thus, it is
likely that the 40% of GLUT4 that was not liberated from
the low speed pellet is of intracellular origin. Hence, the low
speed pellet presumably contains a complement of all cellular
organelles, including unbroken cells, cellular debris, and mem-
branes inside membranes. Becauseof these undesirable char-
acteristics and the fact that the 184,000 x g pellet contained
FIG. 1. Cryosections of rat soleusmuscle, labeled for
GLUT4 (10n m gold in A a n d B, 15 nm in C). Section C was
additionally labeled for rat albumin (5 nm gold).A, basal cell in which
labeled structures a t one side of the golgi cisternae (g) probably
represent the TGR. Small GLUT4-positive elements are grouped
around a large vacuole ( u ) . A small vesicle near the cell surface is
also labeled (arrow),but gold particles are rarely associated with the
plasma membrane itself (arrowheads). n, nucleus; m, mitochondrion.
B, stimulated cell with obvious labeling of the cell surface (arrow-
heads) and of some small vesicles (arrows).z, Z-line of the myofibrils.
C, stimulated cell in which the plasma membrane is not visible due
to oblique sectioning. However, GLUT4 labeling of the cell border,
indicated by the collagen fibers and albumin labeling, is evident
(arrowheads). Tubulo-vesicular elements labeled for GLUT4 are
sometimes associated with vacuoles ( u ) containing rat albumin. Cal-
ibration bar is equal to 200 nm.
the majority of muscle GLUT4 as well as a complement of
markers characteristic of the sarcolemma, sarcoplasmicretic-
ulum, mitochondria, and transverse tubules, further studies
were performed using the 184,000 X g fraction as starting
material.
Vesicle Immunoadsorptwn-Wehavepreviouslyshown
that themonoclonal antibody 1F8 is specific for a cytoplasmic
epitope of GLUT4.3 This antibody was coupled to magnetic
beads conjugated with sheep anti-mouse IgG and incubated
with the resuspended 184,000 X g pellet in the presence of
100 mM NaC1. Maximal binding of IRGTV wasobserved
using 2 x lo5 beadslpg of protein. At this ratio of beads to
membrane protein, the nonspecific binding of GLUT4 to the
beads was <5%. Thus, all further immunoadsorptions were
performed for 240 min at 4 "C with 2 x lo5 beadslpg of
184,000 X g protein.
To analyze the morphology of the IRGTV, freeze fracture
electron microscopy was performedon immunoadsorbed ves-
icles. As shown in Fig. 3,b and c the immunoadsorbed vesicles
1F8 immunoblots a fusion protein expressing the last 25 amino
acids encompassing the carboxy terminus of GLUT4 (Robert Piper,
unpublished data).
 GLUT4 in Muscle 6281

FIG. 2. Section of basal soleus muscle labeled for GLUT4

(15 nm gold) and rat albumin (5 nm gold). The section grazes
the surface of a myofibril, giving a fortuitousview of the surrounding
triads and sarcoplasmic reticulum (SR). Rat albumin delineates the
transverse tubules which appear in isolation (small arrowheads) as
well as in association with sarcoplasmic reticular elements to form
the triads (large arrowheads). The SR is the branched system between
the triads (asterisks). As is usual in cryosections the SR membranes
are weakly visible, but no GLUT4 labeling isapparent.GLUT4

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labeling is present on small vesicles (arrows). z,Z-line; m, mitochon-
drion. Calibration bar is equal to 200 nm.

TABLE I
Subcellularfractionation of basal rat skeletal muscle
Skeletal muscle from overnight fasted rats was rapidly frozen in
situ, powdered,homogenized, and centrifuged at 3,000 X g. The
supernatant was centrifuged a t 184,000X g. Total protein and specific
organelle markers were quantified in the homogenate, the 3,000 X g
pellet, and the 184,000 X g pellet. The protein yield for each pellet
was calculated asthe percentage of proteinin the homogenate. FIG. 3. Electron micrographs of intracellular vesicles con-
Subcellular markers were determined by immunoblotting equal taining GLUT4. Magnetic beads (2 X 10’) were incubated in the
amounts of protein from the homogenate, 3,000 X g, and 184,000 X g absence (a) or presence ( b and c) of 1F8, washed, and incubated with
pellets. These values were quantified by scanning densitometry,mul- the 184,000 X g fraction (100 pg of protein) as described under
tiplied by the total protein/fraction, and expressed as apercentage of “Experimental Procedures.” Beads were then fixed in 0.1 M phosphate
that observed in the homogenate to calculate the yield. The recovery buffer, pH 7.4, containing 2% paraformaldehyde and 0.2% glutaral-
of markers ranged from70-93%. Enrichment of markersin the dehyde for freeze fracture electron microscopy (“Experimental Pro-
184,000 X g fraction was calculated relative to thehomogenate. Each cedures’’). Calibration bar is equal to 200 nm.
value represents the mean f S.E. of two to four different experiments.
3,000 x g 184,000 x g
efficiency of GLUT4 from the 184,000 x g fraction did not
Yield Yield Fold enrichment
reflect limiting amounts of antibody or magnetic beads during
% % immunoisolation because the residual GLUT4 could not be
Protein 46 f 3 8f1 immunoadsorbed following re-incubation with fresh 1F8
GLUT4 3825 55&3 7.7 beads (data notshown).
GLUTl 52f8 18f6 2.2 Of the different markers analyzed, GLUT4 was the only
Na,K-ATPase 65 f 4 19 f 3 2.4
Ca*+-ATPase 55f5 21f4 2.6 protein enriched in the muscle IRGTV (Fig. 4). Na,K-ATP-
tt28 45f4 26f2 3.2 ase, a marker for the sarcolemma, Ca2+-ATPase, a marker for
the sarcoplasmic reticulum, SDH, a mitochondrial marker,
were small vesicles of average size 40-100 nm in diameter. tt28, a marker for the transverse tubules, and GLUTl were
This corresponds with the size range of the major peak of all below the limit of detectability in the immunoisolated
GLUT4 vesicles as isolated by S-1000 gel filtration (see be- fraction (Fig. 4). Similarly, 5’-nucleotidase, a marker for the
low). There was no elaborate coatstructure, such as a clathrin sarcolemma, andthe insulin-like growth factor(IGF) 11/
lattice, associated with the immunoadsorbed vesicles. As mannose6-phosphate receptor, amarker for the receptor
shown in Fig. 3a, very few vesicles were attached tobeads not recycling pathway, were also below the limit of detectability
conjugated with 1F8, indicating the specificity of this tech- in theimmunoisolated IRGTV fraction(data notshown). For
nique for isolating subcellular organelles. each of these markers, we obtained a quantitativerecovery in
The non-adsorbed material was concentrated by centrifu- the non-adsorbed fraction. While we cannot exclude the pos-
gation, and this fraction together with the fraction immu- sibility that the IRGTV contains a small amount of these
noadsorbed to thebeads were immunoblotted with antibodies markers, these data taken together with the immunocyto-
specific for different markers (Fig. 4). GLUT4 was purified chemistry indicate that under basal conditions the majority
40-fold over the 184,000 X g fraction which is equivalent to a of GLUT4 inmuscle is not present in the sarcolemma, trans-
300-fold purification from the tissue homogenate. The yield verse tubules, sarcoplasmic reticulum, and mitochondria. It is
of GLUT4 in the immunoadsorbed fraction was 80 f 3% ( n noteworthy that of the markers examined GLUT4 had the
= 8) of the initial 184,000 X g fraction. This immunoisolation highest nonspecific binding to magnetic beads (Fig. 4). This
6282 GLUT4 in Muscle
in a size range (40-100-nm diameter) consistent with that
observed in electron micrographs of cryosectioned muscle
GLUT4
(Figs. 1and 2) and immunoadsorbed IRGTV (Fig. 3). Markers
for the sarcolemma (Na/K-ATPase), sarcoplasmic reticulum
GLUT1 (Ca2+-ATPase), transverse tubules(tt28), and GLUTleluted
in the column void volume (Fig. 5). This indicates that, with
muscle homogenization, these organelles are dispersed into
Ca ATPase particles larger than 300 nm, the exclusion limit of Sephacryl
s-1000.
The two GLUT4 peaksresolved by S-1000 chromatography
Na/K ATPase were further characterized by immunoadsorption. Fractions
corresponding to the voidvolume(45-54 ml) or the major
SDH GLUT4 peak (78-96 ml) were pooled, concentrated by cen-
trifugation, and incubated with magnetic beads coated with
1F8. The amount immunoadsorbed from the voidvolume
n2a represented 40% of the total GLUT4 in thepeak while more
than 90% of GLUT4 in the 78-96 ml peak resolved by the
column was immunoadsorbed (n = 2, data not shown). Nei-
FIG. 4. Characterization of the immunoisolated GLUT4- ther of the immunoadsorbed fractions contained detectable
containing vesicles from skeletal muscle. 1F8-coupled or control amounts of the other markers shown in Fig. 4. These data
magnetic beads were incubated with the 184,000 X g fraction (100 pg indicate that thenon-immunoadsorbed GLUT4 inthe 184,000
of protein) asdescribed under “ExperimentalProcedures.” Beads ( B ) X gfraction (Fig. 4) correspond to particles of >300-nm
and the non-adsorbed supernatant ( S ) were subjected to sodium diameter. These particles may represent aggregated, en-
dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblot-

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ted with antibodies specific for GLUT4, GLUT1, the Na/K-ATPase, trapped, or introverted vesicles. Some of the small TV ele-
Ca2+-ATPase,succinate dehydrogenase ( S D H ) , and tt28. The data ments may have also remained associated with other larger
shown are representative of four separate experiments. particles under these conditions. The non-immunoadsorbed
GLUT4 particles at the void volume, which constituted only
15% of the total GLUT4 in the 184,000 x g fraction may
represent low levels of GLUT4 in surface membranes, poorly
homogenized T V elements,GLUT4 in large intracellular
structures such as the vacuoles observed in Fig. 1 or IRGTV
trapped inside larger membrane structures.
Effects of Insulin-Subcellular fractionationstudies in
white adipocytes (3) and quantitative immunocytochemistry
in brown adipocytes (8) and cardiac muscle (9) indicate that
200, maximal insulin stimulation causes a 40-50% reduction in
7 GLUT I I the amountof GLUT4 in theIRGTV commensurate with its
Na’/K“ATPase
w 100 translocation to the cell surface. Thus, we reasoned that an
>
- 0 important criterion for referring to the IRGTV as the intra-
C 45 $0 75 90 105 I20 cellular compartment of GLUT4 in muscle would be to dem-
4
-I
200. onstrate a decrease in GLUT4 levels in this fraction. Studies
were performed using muscle from overnight-fasted, anesthe-
w 100
LT tized rats thatwere infused with either saline or a maximally
0 effective dose of insulin (70 milliunits. kg”.min”) and glu-
45 60 75 90 105 120
cose (30 mg. kg” .min”) to maintain euglycemia. Muscles
Elution Volume (ml) were rapidly frozen and fractionated as above. The distribu-
FIG.5. Purification of GLUT4-containingvesicles using gel tion of GLUT4 and theNa,K-ATPase between the 3,000 X g
filtration chromatography. A column of Sephacryl S-1000 (1.6 X and 184,000 X g pellets is shown in Fig. 6. There was no
65 cm) was loaded with 5 mg of protein from the 184,000 X g fraction change in thedistribution of the Na,K-ATPase between these
and eluted at 4 “C with a buffer containing 20 mM HEPES, 1 mM two fractions with added insulin, indicating that the distri-
EDTA, 250 mM sucrose, and 0.02% sodium azide, pH 7.4. Aliquots of
each fraction (3ml) were assayed for total protein andimmunoblotted bution of surface membranes between these fractions is un-
for the insulin-regulatable glucose transporter (ZRGT) GLUT4, altered with insulintreatment. GLUT4 was decreased by 35%
GLUT1, Na/K-ATPase, tt28, and Ca2+-ATPase.Arrows denote the in the 184,000 X g fraction with insulin, but there was no
elution peak for particle standards of known diameter (150, 59 nm). difference in GLUT4 levels in the low speed pellet between
control and insulin-treated animals. Fractionation of the
phenomenon has also been noted usingother matrixes. Thus, 184,000 X gpelletson the S-1000 column again revealed
the IRGTV appear to have unique adherent properties that GLUT4 in the void volume and in the small particle peak
give rise to thiseffect. (Fig. 7). Quantitation of these peaks indicated that insulin
Gel Filtration Chromatography-Further studies using gel reduced the small particulate GLUT4(78-96 ml) by 40%and
filtration chromatography also indicate that the majority of caused a slight increase in the void peak of GLUT4 (45-54
GLUT4 could be separated from the sarcolemma, transverse ml). Collectively, these dataindicate that thefraction we have
tubules, and sarcoplasmic reticulum in nonstimulated muscle. referred to as the IRGTV, which is either peak 2 on the S-
The 184,000 x g fraction from skeletal muscle was fraction- 1000 column or the 184,000 X g-immunoadsorbed fraction
ated on a Sephacryls-1000 column (Fig. 5). Over 70% of the corresponds to theintracellular compartmentof GLUT4. This
GLUT4-containingparticles inthe 184,000 x g fraction eluted percent reduction corresponds well with the effect of insulin
 GLUT4 in Muscle
INSULIN
- + - +
-
Na / K -ATPase
GLUT4
3K 184K
FIG. 6. Effect of insulin on the subcellular distribution of
GLUT4 in skeletal muscle. Overnight-fasted rats were anesthe-
tized and infused with insulin (70 milliunits. kg" .min") and glucose
(30 mg.kg".min"), or saline for 1 h before skeletal muscle was
frozen, homogenized, and fractionated by differential centrifugation.
Aliquots of the 3,000 X g fraction (50 pg) and the184,000 X g fraction
(10 pg), proportionate to therelative yield of these fractions from the
muscle homogenate, were immunoblotted with antibodies specific for
the Na/K-ATPase (above) and GLUT4 (below).
Elution Volume (ml)
FIG. 7 . Gel filtration chromatography of muscle fractions
from basal and insulin-treated rats. Overnight-fasted rats were
anesthetized and infused with insulin (70 milliunits. kg". min") and
glucose (30 mg. kg". min") or saline for 1h. The 184,000 X g fractions
were isolated as described under"Experimental Procedures," and
equal amounts of protein (5 mg) from basal (0)and insulin-treated
(W)rats were subjected to S-1000 chromatography. Fractions were
collected and immunoblotted for GLUT4.
on the intracellular GLUT4 compartment, as revealed by
immunocytochemistry (8).
Whereas an insulin-dependent decrease inGLUT4 was
evident in the 184,000 x g fraction, we did not observe a
parallel increase in the 3,000 x g pellet. This is likely due to
the ratio of cell surface membranes to IRGTV in thisfraction.
Because there is virtually noGLUT4 present at the cell
surface of basal cells (Fig. 1)the GLUT4 present in the3,000
X g pellet from basal muscle (-40% of total) mustarise from
the IRGTV. Thus, the GLUT4 levels in this fraction from
insulin-treated muscle willreflect a combinationof decreased
GLUT4 in the IRGTV as well as an increase in the plasma
membrane component. Based on our calculation of IRGTV/
plasma membrane in thisfraction we estimate thatwe would
have observed a t most a 20-30% increase in GLUT4ievels in
this fraction with insulin. Therefore, the absence of a change
in GLUT4 with insulin in thisfraction is not surprising.
Polypeptide Composition of the IRGTV-The polypeptide
composition of the immunoisolated vesicles from skeletal
muscle is shown in Fig. 8. Cellulose coupled to 1F8, but not
incubated with the skeletal muscle fraction (lane I F 8 ) , ex-
hibits a set of bands derived from the activation and coupling
process. 1F8 coupled and control cellulose (lanes labeled IP
and CTL, respectively) was incubated with the 184,000 x g
muscle fraction. The subset of bands which are common to
both the IP and CTL lanes represent proteins that adhere
6283
M r n 10 ' 4
205
4
21
14
Downloaded from www.jbc.org by on August 26, 2008
FIG. 8. Silver stain of GLUT4-containing vesicles from rat
skeletal muscle. Cellulose coupled with 1F8, but notincubated with
the muscle fraction, reveals the protein constituents of the immuno-
isolation substrate ( l F 8 ) .Cellulose coupled with 1F8 (ZP)
or with an
irrelevant antibody (CTL) was incubated with the 184,000 X g muscle
fraction in 1 ml of PBS containing 1 mg of fish gelatin for 3 h a t
4 "C, then washed with 1 M sodium chloride followed by PBS. A
common subset of nonspecifically adsorbed proteins is observed in
the ZP and CTL lanes. The protein constituents of the specifically
adsorbed IRGTV in the ZP lane are indicated (arrowheads). The
184,000 X g muscle fractions (184 K ) fraction is also shown. The
samples were run on a 5-20% polyacrylamide gel and stained by the
method of Oakley et al. (22).
nonspecifically to thecellulose fibers. Many of these proteins
are also visible in the 184,000 X g starting material (see lane
184 K ) . The protein bands which are unique to the IP lane
(indicated by arrowheads) are due to the specific immunoad-
sorption of the IRGTV. Many of these proteins are highly
enriched in the IRGTV fraction as they are notvisible in the
184 K starting material. Toquantitatethe abundance of
GLUT4 in the muscle IRGTV fraction, the immunoadsorbed
fraction from muscle was immunoblotted with anti-GLUT4
antisera on the same filter as a low density microsome stand-
ard from rat adipocytes containing aknown amount of trans-
porter as determined by glucose-displaceable cytochalasin B
binding (Fig. 9). Assuming that GLUT4 is the major trans-
porter species in this low density microsome fraction (11)and
that GLUT4 has an average molecular mass of45 kDa,
GLUT4 constitutes 5% of the total IRGTV protein.
DISCUSSION
In the present studies we have used immunocytochemical
and biochemical techniques to localize GLUT4 inrat skeletal
muscle. In agreement with previous studies in other insulin-
responsive tissues (1-13), GLUT4 underwent astimulus-
dependent translocation to thecell surface. However, a strik-
ing observation from these studies was the virtual absence of
GLUT4 from the cell surface of non-stimulated muscle. This
is an important observation because, as is the case for white
adipocytes,2brown adipocytes (8) and cardiac muscle (9), the
6284 GLUT4 in Muscle
LDM GT V tein that is found in the TGR.These data together with the
fact that GLUT4 constitutes a high percentage of the vesicle
GLUT4
protein suggests that GLUT4 may be sequestered within a
subcompartment of the TGR. This targeting is very specific
toGLUT4 because another glucose transporter isoform
.25 .50 1.0 2.0 .04 .08 (GLUTl), which shows considerable sequence homology with
GLUT4 (65%), is not present to any large extent in this
Protein (fig)
compartment (32). The TGR is known to have an important
FIG.9. Quantitation of GLUT4 levels in the IRGTV. function in proteinsorting both in terms of newly synthesized
GLUT4-containing vesicles were immunoisolated from muscle and
immunoblotted together with different amounts (0.25-2.0 pg of pro- proteins and recycling molecules. However, the molecular
tein) of a GLUT4 standard (low density microsome ( L D M ) fraction mechanisms by which this sortingis mediated are not under-
from rat adipocytes containing a known concentration of transporter stood.
as determined by cytochalasin B binding). The immunoreactive band The deep etch freeze fracture images of the GLUT4-con-
corresponding to GLUT4 was quantitated in each fraction and used taining vesicles indicate that they constitute small uniform
to estimate the amount of transporter/pg of IRGTV protein. It was particles of similar size to thatpreviously described for TGR-
assumed for the purpose of this estimation that GLUT4 is the major
transporter species in rat adipocyte LDM (10). A M,of 45,000 for derived transport vesicles that play a role in regulating exo-
GLUT4 was used to estimate GLUT4mass. cytic membrane movement (33-35). We have performed sim-
ilar analyses on IRGTV isolated from cardiac muscle, white
magnitude of the stimulation-dependent increase in GLUT4 adipocytes, and brown adipocytes4 and theparticles are iden-
at the cell surface is extremely large and certainly adequate tical to that shown in Fig. 3. In all cases, there is no visible
to account for the associated increase in glucose transport coat structure associated with the vesicles. It was possible,
activity. In contrast,subcellular fractionation studies in mus- however, to identify other molecules present in these vesicles
cle have documented a variable degree of GLUT4 transloca- by silver staining. A number of abundant proteins other than
tion, usually on the order of %"-fold (5-7). The low and/or GLUT4 are present in the IRGTV. These include proteins of

Downloaded from www.jbc.org by on August 26, 2008

variable amount of GLUT4 translocation occurring in these molecular mass -20, 45, 70, 180 and 220 kDa. The 180-kDa
studies is due to high levels of GLUT4 in the plasma mem- protein is probably not clathrinbecause of the absence of any
brane fraction isolated from non-stimulated muscle. In view coat from the electron micrograph images. Furthermore, to
of the discrepancy between GLUT4 translocationand glucose obtain these purified vesicles, conditions were used (1M NaCl
transport in these studies, it has been concluded that there wash) which would be expected to dissociate clathrin. It will
must be an additional effect of stimulation termed "intrinsic be important to identify some of these proteins onthe premise
activation" to account for this difference. Based on our stud- that they may play a role in sequestration of GLUT4. In
addition, these proteins maybe involved in the signaling
ies, we propose that translocation of GLUT4 is the major
mechanisms by which insulin recruits GLUT4 into the en-
mechanism for increasing glucose transport activity in skele-
dosomal recycling pathway. Signaling molecules that may be
tal muscle and that references to the intrinsic activation of
of interest include phosphoproteins (36) and proteins referred
glucose transporters in muscle should be interpreted with
to asras-related GTP-binding proteins (37). We have identi-
some degree of skepticism.
fied several phosphoproteins in the IRGTV from adipocytes
An important question concerning the regulation of GLUT4
(36). In addition, we have preliminary evidence that several
in insulin-sensitive cells is the nature of the intracellular
GTP-binding proteins (-20 kDa) reside in the IRGTV iso-
compartment in which the molecule resides under basal con- lated from skeletal muscle and adipocytes using ["PIGTP
ditions. The immunocytochemical data are most informative overlay (data not shown). This is notable because it has been
showing that GLUT4 is enriched in the TGR and near en- shown that GTPrS stimulates GLUT4 translocation in per-
docytic vacuoles close to theTGR. Indeed, the localization of meabilized adipocytes (38). We are currently characterizing
GLUT4 in skeletal muscle is precisely the same as observed these molecules and otherproteins specific to the IRGTV to
previously in white adipocytes,' brown adipocytes (8), and determine if they areregulated similarly to GLUT4 inskeletal
cardiac muscle (9). In addition, we have observed similar muscle and othercell types.
localization of GLUT4 when expressed in non-insulin cell A major function of the IRGTV is its regulation by insulin.
types using DNA transfection (31), suggesting that the ma- We have shown previously that in adipocytes (8) and cardiac
chinery required for this targeting is expressed in many dif- muscle (9), insulin stimulation results in a marked increase
ferent cell types. In each of these cell types, including skeletal in cell surface GLUT4 and an approximate 50% reduction in
muscle, GLUT4 is not localized to endosomes under non- GLUT4 labeling within the TV elements. Insulin appears to
stimulated conditions, whereas in the presence of insulin have a similar effect on GLUT4 in skeletal muscle. While
GLUT4 labeling is increased in both plasma membranes and further study is required to quantify the magnitude of this
endosomes. effect, as well as the relative contributions of insulin and
It is critical to determine how GLUT4-containing vesicles exercise, the immunocytochemical data implicate transloca-
are mobilized by insulin or exercise stimulation. The machin- tion asbeing a major mechanism for regulating glucose trans-
ery required for this process may also be found inthe IRGTV. port in skeletal muscle. However, using subcellular fraction-
For this reason we have attempted to biochemically charac- ation it hasbeen difficult to ascertain the degree of GLUT4
terize this compartment. Two fractionation techniques were translocation to the cell surface in skeletal muscle. This is
used to isolate and purify this compartment from skeletal due to the difficulty in resolving intracellular membranes
muscle. The immunoisolated IRGTV do not contain appre- from cell surface membranes. This was also our experience in
ciable levels of markers for the sarcolemma, transverse tu- the presentstudies. However, using the additional procedures
bules, sarcoplasmic reticulum, and mitochondria as expected described here it was possible to isolate the intracellular
from the immunocytochemistry. Furthermore, the IRGTV membranes containing GLUT4 in reasonable yield and purity.
isolated from muscle (data not shown) or adipocytes (11) do As a result, we observed a 40% decrease in GLUT4 levels in
not contain appreciable levels of the mannose 6-phosphate
receptor (insulin-like growth factor I1 receptor), another pro- K. J. Rodnick, unpublished observation.
 GLUT4 in Muscle 6285
this fraction following insulin stimulation (Figs. 6 and 7). An B. E., Ruoho, A. E., and Pilch, P. F. (1989) J. Bioi. Chem. 2 6 4 ,
effect of this magnitude is consistent with that observed 12358-12363
previously in a variety of insulin-sensitive tissues following 12. Piper, R. C., Hess, L. J., and James, D. E. (1991) Am. J. Physiol.
2 6 0 , C570-C580
(39 5-9). We Observed a modest 13. James, D. E., Lederman, L., and Pilch, P. F. (1987) J. Biol. Chem.
dependent increase in GLUT4 levels in membrane fractions 262,11817-11824
enriched in cell surface markers (Fig. 7). However, in view of 14. Friedman, J. E., Dudek, R. W., Whitehead, D. S., Downes, D. L.,
the contamination ofthese fractions with GLUT4 under basal Frisell, W. R., Caro, J. F., and Dohm, G. L. (1991) Diabetes 4 0 ,
150-154
it is not possible to equate these data with 15. DeFronzo, R.A., Jacot, E., Jequier, E., Maeder, E., Wahren, J.,
action in the intact cell. Thus, in the future it is likely that and Felber, J. P. (1981) Diabetes 3 0 , 1000-1007
immunocflochemist~Will be the best approach to accurately 16. Curtis-Prior, P. B., Trethewey, J., Stewart,G. A., and Hanley, T.
localize GLUT4 and quantify the degree of GLUT4 translo- (1969) Diabetologia 5 , 384-391
cation in skeletal muscle. 17. James, D. E., Jenkins, A. B., and Kraegen, E. W. (1985) Am. J.
Physiol. 2 4 8 , E567-E574
18. James, D. E., Strube, M., and Mueckler, M. (1989) Nature 3 3 8 ,
Acknowledgments-We are grateful to Dr. John Heuser for per- 83-87
forming the electron microscopy studies on immunodsorbed IRGTV 19. Jorgensen, A. O., Arnold, W., Shen, A. C.-Y., Yuan, S., Gaver,
bound to magnetic beads. We are also grateful to Robert Piper and M., and Campbell, K. (1990) J. Cell. Biol. 110, 1173-1185
Dr. John Holloszy for their helpful COmmntS during the COurSe of 20. Reynolds, J. A., Nozaki, y., and Tanford, C. (1983) Anal.
these studies. Finally, we thank the following individuals for gener- Biochem. 130,471-474
ously providing antisera for these experiments: Drs. John Lawrence 21. Ackers, G. K. (1967) J , ~ i ~chern,
l . 2 4 2 , 3237-3238
and Robert Mercer, Washington University School of Medicine; Dr. 22. Hales, C. N.,and Woodhead, J. S. (1980) Methods Enzymol. 70,
Kevin Campbell, University of Iowa; Dr. Paul Luzio, Cambridge 334-335
University, U.K.; Dr. Rob Baxter, Sydney University, Australia; and 23. Oakley, B.R., Kirsch, D.R., and Morris, N. R. (1980) Anal.
Dr. Mario Rosenblatt from Santiago, Chile. Biochem. 105,361-363
24. Heuser, J. E. (1983) J. Mol. Biol. 169, 155-195
25. Heuser, J. E. (1989) J. Cell. Biol. 109, 1457-1466
26. Heuser, J. E. (1980) J. Cell. Biol. 8 4 , 560-583

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