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and -
with Glucose Transporter-4-Containing Vesicles in Primary Rat Adipocytes
Department of Cell Biology, Parke-Davis Pharmaceutical Research Division, Warner-Lambert Company, Ann Arbor, Michigan 48105
Address all correspondence and requests for reprints to: C. C. Mastick, Parke-Davis Pharmaceutical Research Division, Warner-Lambert Company, Ann Arbor, Michigan 48105. E-mail: masticc{at}aa.wl.com
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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-SNAP,
and 
-SNAP in primary rat adipocytes were determined. A large
fraction of the NSF and SNAPs were associated with intracellular
membranes, distributed between the low-density microsomes (LDM) and
high-density microsomes. Very little of the NSF and SNAPs were
associated with the plasma membrane fraction. This distribution did not
change after insulin stimulation. Approximately 75% of the NSF and
SNAPs in the LDM fraction were coimmunoprecipitated with 85% of the
GLUT4 and 60% of the vesicle associated membrane proteins (VAMPs;
synaptobrevins) VAMP-2 and cellubrevin in anti-GLUT4 immunoadsorptions.
In contrast to NSF and the SNAPs, the ß-coatomer protein (ß-COP)
found in the LDM fraction was excluded from GLUT4 vesicles. When LDM
fractions were solubilized with Thesit (octaethylene glycol dodecyl
ether) or Triton X-100, approximately 40% of the -SNAP was
colocalized with NSF on glycerol gradients in large (
20S),
ATP-sensitive complexes. VAMP-2 and cellubrevin are concentrated in the
LDM fractions and in GLUT4 vesicles; both were excluded from these
complexes. These data suggest that the steady state association of NSF
and the SNAPs with GLUT4 vesicles and cell membranes is independent of
the formation of fusion complexes. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Recently, a role for the VAMPs in vesicle targeting and fusion to the
plasma membrane has been proposed (16–18, reviewed in Refs. 19–22).
The VAMPs form complexes with two plasma membrane-associated proteins,
syntaxin and synaptosome-associated protein of 25 kDa (SNAP-25) (17, 18, 23). This complex of three proteins is very stable (23) and forms a
high affinity binding site for several soluble, cytosolic proteins
called the soluble N-ethylmaleimide sensitive fusion protein
(NSF) attachment proteins (SNAPs) (three isoforms: , ß, and ;
no relation to SNAP-25) (17, 18). The SNAPS are required for the
association of NSF with membranes (24, 25, 26). Although its role is
incompletely understood, NSF is required for the fusion of lipid
bilayers in most vesicle fusion events in eukaryotic cells, in a
process that requires ATP hydrolysis (reviewed in Refs. 19, 21, 22,
27–29). The VAMPs, syntaxins, SNAPs, and NSF can bind to form a 20S
complex, which is dissociated on ATP hydrolysis. Based on these data,
the VAMPs were termed the vesicle associated receptors for the SNAPs
and NSF (v-SNAREs), and the syntaxins and SNAP-25 were termed the
target membrane receptors for these proteins (t-SNAREs). A
v-SNARE/t-SNARE complex was thought to be required for the association
of NSF and SNAP with membranes. However, this idea has recently been
challenged by Mayer et al. (30) and Colombo et
al. (31), who found that NSF and -SNAP function at steps
preceding vesicle docking and fusion. Formation of complexes between
specific pairs of v-SNAREs and t-SNAREs was suggested to be one
mechanism regulating the fusion of vesicles to the proper target
membranes. It was further proposed that regulation of the formation of
the VAMP/syntaxin fusion complexes would be a mechanism to regulate the
fusion of vesicles to the plasma membrane (17, 21, 22).
To elucidate the role of fusion complexes in the regulation of the
trafficking of GLUT4 in primary rat adipocytes, we investigated the
effects of insulin on the association of endogenous NSF, -SNAP, and
-SNAP with membrane fractions and GLUT4 vesicles. The NSF and SNAPs
are highly enriched in GLUT4 vesicles and low-density microsomes (LDM)
under steady state conditions, and this association is independent of
insulin stimulation. Very little of the NSF and SNAPs are associated
with the plasma membrane (PM) fraction. Consistent with the findings of
Mayer et al. (30) and Colombo et al. (31), these
data suggest that complexes of NSF and SNAPs can associate with
adipocyte membranes independently of the formation of fusion complexes.
This association does not appear to involve the v-SNAREs VAMP-2 or
cellubrevin.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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S
were purchased from Sigma (St. Louis, MO). Octaethylene glycol dodecyl
ether (C12E8; Thesit) and Combithek
chromatography calibration standards were purchased from Boehringer
Mannheim (Indianapolis, IN). Prestained molecular weight markers (high
range) and horseradish peroxidase conjugated-goat antimouse and goat
antirabbit IgG antibodies were purchased from GIBCO-BRL (Gaithersburg,
MD). All other reagents were purchased from Sigma. Protein
concentrations were determined using the BCA protein determination kit
from Pierce (Rockford, IL).
Antibodies
Affinity-purified rabbit antibodies against the carboxy-terminal
peptide of GLUT4 (amino acid residues 491–509; anti-GLUT4) were a
generous gift from Dr. Gustav Lienhard (32). Affinity-purified rabbit
antibodies against histidine-tagged recombinant NSF, -SNAP, and
-SNAP were a generous gift from Dr. Thomas Söllner (33). Mouse
monoclonal anti-ß-coatomer protein (COP) antibody maD was a generous
gift from Dr. Thomas Kreis (34). Affinity-purified rabbit antibodies
against a peptide from the hydrophilic core region of both rat brain
VAMP-1 and -2 (amino acid residues 55–70 of VAMP-1) were a generous
gift from Dr. William Trimble (8).
Cell fractionation
Adipocytes were prepared from the epididimal fat pads of 24
Sprague-Dawley rats by collagenase digestion as described (35). To
compare basal and insulin-treated cells, half of the cells were treated
with 10 nM insulin at 37 C for 30 min. The cells were
washed and homogenized in 255 mM sucrose (8.4%), 20
mM HEPES, 1 mM EDTA, pH 7.4 (homogenization
buffer), and fractionated by differential centrifugation into LDM, high
density microsomes (HDM), PM, mitochondria/nuclei, and cytosol as
described (36). Briefly, the homogenates were centrifuged at
16,000 x g to separate the PM and mitochondria/nuclei
(pellet) from the HDM, LDM, and cytosol (supernatant). The pellet was
resuspended in homogenization buffer, and the PM was resolved from the
mitochondria and nuclei using a 33% wt/vol sucrose step gradient. The
16,000 x g supernatant was centrifuged at 48,000
x g to prepare the HDM fraction (in pellet), then
centrifuged at 150,000 x g to separate the LDM
(pellet) from the cytosol (supernatant). The membrane pellets were all
resuspended in 2 ml homogenization buffer. Approximately 10 ml of
cytosol were recovered from each condition (12 rats). Samples were
quick frozen in liquid nitrogen and stored at -80 C.
Immunoadsorption of GLUT4 vesicles
The protocol for the isolation of GLUT4 vesicles from the LDM by
immunoadsorption has been described in detail previously (8). Briefly,
NaCl was added to 48,000 x g supernatants (1.3 ml,
derived from the adipocytes of 1 rat, containing about 100 µg of LDM)
to a final concentration of 100 mM, and vesicles
immunoadsorbed with anti-GLUT4 or a nonspecific rabbit IgG bound to the
surface of formaldehyde-fixed S. aureus cells (6 µg
antibody/2 µl S. aureus cells). The adsorbed proteins were
sequentially eluted first with 100 µl 1%
C12E8 in homogenization buffer/100
mM NaCl to elute vesicle proteins, followed by 133 µl SDS
sample buffer containing 8 M urea to elute antibodies and
proteins bound to the antibodies. LDM was isolated from the original
48,000 x g supernatants and from the supernatants
after immunoadsorption by centrifugation at 150,000 x
g.
Velocity and sucrose gradient sedimentation
Glycerol gradients (1.2 ml) were prepared by layering 300 µl
each 35%, 25%, 15%, and 5% glycerol in 20 mM HEPES, 1
mM EDTA, 100 mM NaCl, 1 mM
dithiothreitol (DTT), 0.5% C12E8, and 0.5
mM Mg-ATP or ATP-S in 2-ml tubes. Gradients were allowed
to equilibrate for 1 h at room temperature before use. LDM
fractions were prepared as described above from basal or
insulin-stimulated adipocytes (12 rats/condition), washed once with
homogenization buffer, and resuspended in 500 µl homogenization
buffer/100 mM NaCl. To solubilize the membranes, 50 µl of
the LDM fraction (100–300 µg protein) were diluted 1:1 with 50 µl
solubilization buffer (20 mM HEPES, 1 mM EDTA,
100 mM NaCl, 1% C12E8, 2
mM DTT, and 1 mM Mg-ATP or ATP-S), and the
samples incubated for 1 h on ice with frequent mixing. This
incubation was necessary because immediately after addition of
C12E8 to the LDM samples, the NSF, -SNAP,
and -SNAP were in large complexes that pelleted at relatively low
speeds (16,000 x g) and were not retained on the
gradients (data not shown). The VAMPs were excluded from these
complexes. These large complexes were not observed when proteins in
isolated GLUT4 vesicles were solubilized with
C12E8 (see Immunoadsorption of GLUT4
Vesicles). The solubilized samples were centrifuged at 16,000
x g in a microfuge for 5 min to remove insoluble
material.
Sucrose gradients (1.2 ml) were prepared by layering 300 µl each
20%, 15%, 10%, and 5% sucrose in 20 mM HEPES, 1
mM EDTA, 100 mM NaCl, 0.5% Triton X-100 in
2-ml tubes. Gradients were allowed to equilibrate overnight at 4 C. The
LDM samples for the sucrose gradients (75 µg in homogenization
buffer) were diluted to 20 mM HEPES, 1 mM EDTA,
100 mM NaCl, 1% Triton X-100, 2.2% sucrose, and incubated
1 h at 4 C. No difference in the migration of the VAMPs was
observed when the LDM were solubilized with
C12E8 instead of Triton X-100, or if ATP or
ATP-S were included in the solubilization buffer (data not
shown).
The solubilized samples were carefully layered on top of the glycerol (100 µl) or sucrose (200 µl) gradients, and the gradients were centrifuged in a TLS-55 swinging bucket rotor (Beckman Instruments, Inc., Palo Alto, CA) at 50,000 rpm (166,000 x g) for 2.5 h (glycerol gradients) or 4 h (sucrose gradients) at 4 C. The gradients were then fractionated into 110-µl (glycerol gradients) or 120-µl (sucrose gradients) fractions from the top of the gradients using a gel loading pipette tip. Thirty microliters of each fraction and 10 µl of the original LDM sample were used for Western blotting. Protein standards were run on parallel gradients.
Gel electrophoresis and immunoblotting
Samples in Laemmli sample buffer (4% SDS, 115 mM
Tris-Cl, pH 6.8, 1 mM EDTA, 10% glycerol, 4 mg/ml
bromophenol blue) with 10 mM DTT were boiled for 2 min,
proteins were separated by SDS-PAGE, and the proteins were transferred
to Immobilon-P membranes (Millipore Corp., Bedford, MA)
electrophoretically for 2 h at 400 mA in 20% methanol, 192
mM glycine, 25 mM Tris, 0.005% SDS. (Samples
for determination of GLUT4 were not boiled.) The membranes were blocked
by incubation for 1 h in TBST (20 mM Tris, pH 7.6, 150
mM NaCl, 0.05% Tween 20, 0.01% NP40, 5% nonfat dried
milk), then incubated overnight with antibody diluted in TBST with 1%
milk (anti-NSF, anti--SNAP, anti--SNAP, 1:500; anti-ß-COP,
1:1000; anti-VAMP, 1 µg/ml; anti-GLUT4, 6 µg/ml). After washing
four times in TBST, the membranes were incubated for 30 min with
horseradish peroxidase-conjugated goat antirabbit IgG antibody or goat
antimouse IgG antibody diluted in blocking buffer (1:3000), washed two
times in TBST and two times in TBS (20 mM Tris, pH 7.6, 150
mM NaCl), and the labeled proteins visualized by the
enhanced chemiluminescence method (Amersham, Arlington Heights,
IL).
Image processing
Autoradiographs were quantified by computer-assisted video
densitometry using the Bio Image system (Imaging Systems, Millipore
Corp., Ann Arbor, MI). Figures of autoradiographs were constructed
using a Microtek 600ZS ScanMaker Scanner (Microtek International,
Hsinchu, Taiwan, R.O.C.) and Adobe Photoshop software (Adobe Systems,
Mountain View, CA).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-SNAP, and -SNAP-SNAP, and -SNAP in these
fractions are shown in Fig. 1
. The two isoforms of VAMP
detected in these fractions are VAMP-2 (18 kDa) and cellubrevin (17
kDa) (14, 15). As has been reported previously (8), the highest
concentrations of GLUT4 and both of the VAMPs in basal cells were in
the LDM (lane 3), with less in the PM fraction (lane 5), and very
little in the cytosol (lane 1), or mitochondria/nuclei fractions (lane
9). A significant amount of GLUT4 was also found in the HDM fraction
(lane 7), but very little of either of the VAMPs were found in this
fraction. In response to insulin there was a decrease of GLUT4, VAMP-2,
and cellubrevin in the LDM (55% and 35%, respectively; lane 4)), and
a concomitant increase of all three of these proteins in the PM (2- to
5-fold; lane 6), indicating translocation (3, 8, 10).
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-SNAP, and -SNAP were compared with
those of GLUT4 and the VAMPs. Although NSF, -SNAP, and -SNAP are
soluble, cytosolic proteins, a majority of these proteins were bound to
membranes under steady state conditions, distributed between the LDM
(lanes 3 and 4) and HDM (lanes 7 and 8). There was very little
association of these proteins with the PM fraction (lanes 5 and 6).
Correcting for the amount of each fraction loaded on the gel (and
assuming equal recovery of the fractions), 1–2% of the NSF, 8% of
the -SNAP, and 38% of the -SNAP were found in the cytosol. The
remaining -SNAP and -SNAP were distributed about equally in the
LDM and HDM fractions, whereas the NSF distributed 1:1:2 in the LDM,
HDM, and mitochondrial/nuclei fractions. Less than 1% of each of these
proteins was found in the PM fraction. In contrast to GLUT4 and the
VAMPs, the distributions of NSF and the SNAPs were not changed by
insulin (compare lanes 1, 3, 5, 7, and 9 with 2, 4, 6, 8, and 10).
Therefore, insulin does not affect the steady state levels of
association of the fusion machinery proteins with membranes.
Localization of NSF, -SNAP, and -SNAP in GLUT4 vesicles
The extent to which NSF, -SNAP, and -SNAP were colocalized
with GLUT4 and the VAMPs in the LDM fraction was assessed by
immunoadsorption of vesicles with antibodies against GLUT4 (Fig. 2). Under conditions in which approximately 85% of the
GLUT4 and 60% of both VAMP-2 and cellubrevin were immunoadsorbed (Fig. 2 and data not shown) (3, 8, 10), 70–80% of the NSF, -SNAP, and
-SNAP were depleted from the LDM fraction (Fig. 2, compare lanes 2
and 3). The NSF, -SNAP, and -SNAP were recovered in the
C12E8 elution from the beads (lane 5). The
GLUT4 was recovered in the SDS elution (lane 7). There was no
adsorption of these proteins to beads loaded with a nonspecific
antibody (lanes 4, 6, and 8). Therefore, a significant fraction of the
NSF and SNAPs associated with the LDM were bound to GLUT4 vesicles.
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shows the recovery of GLUT4, NSF, -SNAP, and
-SNAP in GLUT4 vesicles isolated from LDM from either basal or
insulin-stimulated cells. Insulin decreased the GLUT4 content of the
LDM fraction 48% (data not shown), and the recovery of GLUT4 in the
immunoprecipitates was reduced by 42% after insulin stimulation, as
has been previously reported (3). There was a smaller decrease in the
amount of NSF (12%), -SNAP (30%), and -SNAP (23%) recovered
with the GLUT4 vesicles after insulin stimulation. This decrease was
probably due to the decrease in the total number of GLUT4 vesicles in
this fraction, and not to a direct effect of insulin on the association
of these proteins with GLUT4 vesicles. No change in the levels of NSF
and SNAPs were detected in the LDM fractions with short insulin
treatments (3 and 10 min), when little translocation of GLUT4 was
observed (data not shown). NSF and the SNAPs are found as soluble,
cytoplasmic proteins, and are thought to be released from membranes
after fusion. The fact that NSF and the SNAPs were not translocated
with GLUT4 to the plasma membrane suggests that these peripheral
proteins were released and recycled back to the LDM and GLUT4 vesicles
after fusion. We have previously observed a similar rapid recycling of
the secretory carrier membrane proteins (SCAMP)s from the PM to the LDM
after insulin stimulation (3). Alternatively, NSF and the SNAPs may be
enriched in GLUT4 vesicles that are not mobilized by insulin.
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), with some
association with the LDM, HDM, and mitochondria/nuclei fractions. Very
little of the ß-COP was found in the PM fractions. The steady state
distribution of ß-COP was not affected by insulin. Unlike NSF and the
SNAPs, the ß-COP associated with the LDM did not coimmunoprecipitate
with GLUT4 (Fig. 4B). These data indicate that the GLUT4-containing
vesicles are a specific population of intracellular vesicles.
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-SNAP in LDM form large ATP-sensitive complexes that
exclude the VAMPsS. The solubilized proteins were
resolved on glycerol velocity gradients (Fig. 5). When
the membranes were solubilized in the presence of ATP-S,
approximately 40% of the -SNAPs comigrated with the NSF in large
(20S) complexes (Fig. 5, A and C, fractions 9–12). These complexes
were largely dissociated when the membranes were solubilized in the
presence of hydrolyzable ATP (Fig. 5, B and D, fractions 7–9).
Surprisingly, neither VAMP-2 or cellubrevin were detected in the 20S
complexes (Fig. 5, A and C), even with long exposures of the gels (Fig. 5A). No differences in the distributions of these proteins were seen
when LDM from basal or insulin stimulated cells were used in these
experiments, or when Triton X-100 was used instead of
C12E8 to solubilize membranes (data not
shown).
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) or C12E8 (data
not shown). When the solubilized LDM were resolved on sucrose density
gradients, the cellubrevin and VAMP-2 migrated as expected for proteins
of monomeric molecular masses of 17 and 18 kDa (Fig. 6; compare the
migration of the VAMPs in B with the 12- and 25-kDa
standards in C). These data strongly suggest that neither
cellubrevin or VAMP-2 are part of the SNARE complex involved in the
binding of NSF and the SNAPs to the LDM membranes.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In unstimulated cells, GLUT4 is found almost exclusively in
intracellular vesicles distributed throughout the cytoplasm (2). In
response to insulin, the amount of GLUT4 greatly increases in the
plasma membrane and in early endosomes [ 35- and 5-fold,
respectively; (2)], due in part to a significant increase in the rate
of GLUT4 vesicle fusion to the plasma membrane (38, 39, 40). There is also
an increase in the rate of exocytosis of mannose-6-phosphate receptors,
transferrin receptors, and the secreted protein adipsin after insulin
stimulation. Based on this data, the SNARE hypothesis would predict an
increase in the association of NSF and SNAPs with the PM, LDM, and
GLUT4 vesicles after insulin stimulation because the rate of vesicle
fusion greatly increases. This was not observed, however. A significant
amount of the NSF and SNAPs were associated with intracellular vesicles
(LDM and GLUT4 vesicles) under basal conditions, and no change in this
distribution was observed after insulin stimulation (
Figs. 1–3
). In
addition, almost none of these proteins were bound to the plasma
membrane under either condition, despite a large increase in the rate
of vesicle fusion to these membranes.
The lack of association of the NSF and SNAPs with the PM fraction is
not a lack of detection of this association due to the relative
abundance of membranes in this fraction. In isolated rat adipocytes,
90% of the cell volume is taken up by the fat droplet. Therefore the
ratio of plasma membranes to intracellular membranes is very high in
adipocytes relative to other cell types. The PM fraction largely
excludes contaminating intracellular membranes, and equal amounts of
protein are recovered in the LDM, HDM, and PM fractions. In addition,
the levels of GLUT4 and the VAMPs per microgram of protein are
equivalent in the LDM and PM fractions after insulin stimulation (Fig. 1). Therefore, it was expected that similar amounts of NSF and the
SNAPs would be associated with the PM, LDM, and HDM fractions,
particularly after insulin stimulation.
One explanation for the observed steady state distributions of the NSF
and SNAPs would be that fusion complexes accumulate during the process
of intravesicular fusion, but not during the fusion of vesicles to the
plasma membrane. This would imply that the release of NSF and SNAPs
from the fusion complexes between intracellular vesicles and
GLUT4-containing vesicles is different than the release of NSF and
SNAPs from the VAMP/syntaxin fusion complexes at the PM, although the
mechanisms of fusion are thought to be similar. However, the fact that
insulin had no effect on the steady state distributions of NSF and the
SNAPs strongly suggests that the steady state associations of NSF and
the SNAPs with membranes does not reflect the relative amount of fusion
occurring in these membrane fractions. An alternative explanation for
the data is that association of NSF and the SNAPs with membranes is
independent of the binding of the v-SNAREs to their t-SNAREs, and that
the true fusion complex intermediates containing v-SNAREs, t-SNAREs,
NSF, and SNAPs do not accumulate in cells. This idea is consistent with
data in other cell types; very few v-SNARE/t-SNARE complexes are
detected in cell lysates (22, 23). In addition, Mayer et al.
(30) and Colombo et al. (31) have recently shown that NSF
and -SNAP function at an early step before vesicles bind to their
target membranes, and NSF is bound to undocked endosomes,
clathrin-coated vesicles, and synaptic vesicles (31, 41, 42). The high
degree of colocalization of NSF and the SNAPs with GLUT4 in adipocytes
indicates that under steady state conditions a significant proportion
of the NSF and SNAPs are associated with post-Golgi, endocytic
compartments involved in regulated membrane trafficking in these cells.
Perhaps this association indicates a prefusion intermediate, with these
proteins handed off to the SNARE complexes after the binding of the
vesicles to the plasma membranes.
The colocalization of NSF and the SNAPs with VAMP-2 and cellubrevin in
GLUT4 vesicles suggested that one or both of the VAMPs were part of the
membrane receptor binding the NSF and SNAPs to these membranes.
However, several observations indicate that this is probably not the
case. The levels of NSF and the SNAPs associated with the membrane
fractions is not proportional to the amount of either of the VAMPs in
these fractions. A significant proportion of the membrane-bound NSF and
SNAPs are associated with the HDM, a fraction containing little of
either VAMP (Fig. 1). The levels of VAMP-2 and cellubrevin in the LDM
fraction and the GLUT4 vesicles decrease significantly with insulin
stimulation (Ref. 8 and Fig. 1), whereas there is little effect of
insulin on the association of the NSF and SNAPs with these fractions
(Figs. 1 and 3). In addition, the NSF and SNAPs bound to membranes in
the LDM form large (20S) ATP-sensitive fusion complexes when the
membranes are solubilized with C12E8, however,
both the VAMP-2 and cellubrevin in the LDM are excluded from these
complexes (Fig. 5). These data are consistent with recent experiments
that show that recombinant NSF and SNAPs will not form complexes with
the VAMPs in isolated LDM fractions from adipocytes unless the PM
fraction, which is enriched in the t-SNARE syntaxin (43), is also
included in the reaction (44). These data support the idea that the NSF
and SNAPs in the GLUT4 vesicles and LDM are not binding to
v-SNARE/t-SNARE fusion complexes.
It has been proposed that formation of v-SNARE/t-SNARE fusion
complexes is regulated by the association of the SNAREs with other
proteins such as synaptophysin (VAMP), Munc-18 (syntaxin), and
synaptotagmin (reviewed in 22 . Our preliminary data suggest that
neither VAMP-2 or cellubrevin are associated with any proteins in
complexes that are resistant to Triton X-100 solubilization (Fig. 6)
under conditions that should preserve interactions between the VAMPs
and proteins such as synaptophysin (45). Recently, several isoforms of
syntaxin (43) and Munc-18 (46) have been identified in the plasma
membranes of rat adipocytes. It will be interesting to see whether
insulin regulates the interaction of syntaxin and regulatory proteins.
Determination of the mechanism by which the VAMPs in the intracellular
vesicles are prevented from interacting with their cognate t-SNAREs,
the syntaxins, at the plasma membrane may yield important insights into
the mechanism of regulation of glucose transport by insulin.
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Acknowledgments |
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Received October 3, 1996.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-SNAP
binding site. J Biol Chem 270:2213–2217-SNAP) can precede docking and fusion of yeast
vacuoles. Cell 85:83–94[CrossRef][Medline]
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), 
) in each fraction were determined by densitometry and normalized
to total of each in all fractions of gradient (integrated OD/sum of
integrated ODs). Protein standards cytochrome-C (12.5 kDa, 1.75S),
chymotrypsinogen A (25 kDa, 2.6S), ovalbumin (45 kDa, 3.6S), BSA (68
kDa, 4.2S), aldolase (158 kDa, 7.4S), catalase (240 kDa, 11.3S), and
thyroglobulin (670 kDa, 19.3S) were run on parallel gradients without
DTT and peaked in fractions: 1–3 (1.75S), 2–4 (2.6S), 3–4 (3.6S),
3–5 (4.2S), 4–6 (7.4S), 7–8 (11.3S), and 9–11 (19.3S),
respectively.
), and chymotrypsinogen (25 kDa) and catalase (240 kDa) were run on
another (
).