Download 5 x buffer (50TB 25 7 - American Journal of Physiology

AJP-Endo Articles in PresS. Published on April 9, 2002 as DOI 10.1152/ajpendo.00441.2001
E00441-R1
GLUT-4 containing vesicles are released from membranes by
phospholipase D cleavage of a GPI anchor.
Søren Kristiansen and Erik A. Richter
Copenhagen Muscle Research Centre
Department of Human Physiology
Institute of Exercise and Sports Sciences
University of Copenhagen
13 Universitetsparken
2100 Copenhagen
Denmark
Correspondence: Erik A. Richter
At the above address or
by phone + 45 35 32 16 26
or fax
+ 45 35 32 16 00
or E mail : [email protected]
Running title: Glucose transport in skeletal muscle.
Keywords: Insulin signaling, glucose metabolism, rat skeletal muscle.
Copyright 2002 by the American Physiological Society.
2
ABSTRACT.
We have previously developed a cell-free assay from rat skeletal muscle which displayed
in vitro glucose transporter 4 (GLUT-4) transfer from large to small membrane structures
by the addition of a cytosolic protein fraction. By combining protein fractionation and the
in vitro GLUT-4 transfer assay, we have purified a glycosylphosphatidylinositol
phospholipase D (GPI-PLD) which induces transfer of GLUT-4 from small to large
membranes. The in vitro GLUT-4 transfer was activated and inhibited by suramin and
1,10 phenanthroline (an activator and an inhibitor of GPI PLD activity, respectively).
Furthermore, upon purification of the GLUT-4 transporter protein, the protein displayed
an elution profile where the molecular weight was related to the charge, suggesting the
presence or absence of phosphate. Secondly, by photoaffinity labeling of the purified
GLUT-4 with [125I] 3-(trifluromethyl)-3-(m-[125I]iodophenyl) diazirine both labeled
phosphatidylethanolamine and fatty acids (constituents of a GPI link) were recovered.
Third, by using phase transition of triton X –114, the purified GLUT-4 was found to be
partly detergent resistent, which is a known characteristic of GPI-linked proteins. Fourth,
the purified GLUT-4 protein was recognized by an antibody raised specifically against
GPI links. In conclusion, GLUT-4 containing vesicles may be released from a membrane
compartment by action of a GPI PLD.
3
INTRODUCTION.
The insulin and muscle contraction-responsive glucose transporter 4 (GLUT-4)
containing-vesicles are localized to tubulovesicular elements in the basal state (28). Upon
stimulation, the GLUT-4 containing vesicles undergo translocation to the surface
membrane. Despite extensive scrutiny the upstream insulin and contraction mediated
signaling proteins leading to GLUT-4 translocation are not described in great detail. We
have previously focused on immunoprecipitation of low spin membranes containing
GLUT-4 protein and looked for other protein(s) associated within the membrane
compartment (18). Using this approach, we found that the GLUT-4 protein was
associated with an insulin-insensitive phosphatidylinositol (PI) 4- kinase. Furthermore, in
a recent published paper (17) we also reported that in vivo insulin stimulation resulted in
an increased production of phosphatidic acid in the GLUT-4 immunoprecipitate. Since
phosphatidic acid is a known product of phospholipase D (PLD) action, this suggested to
us that a PLD could be part of the mechanism leading to GLUT-4 mobilization. For a
number of reasons we initially hypothesized that it could be the ADP ribosylation factor
(ARF) sensitive PLD1 (14). Due to the restricted experimental models revealing ongoing
enzymatic reactions in skeletal muscle, we were prompted to develop an in vitro assay in
order to investigate whether ARF1 or 6 and subsequent PLD1 activation could lead to
GLUT-4 translocation (17) which was also suggested by Emoto et al. (9). In brief, we
reported that adding a cytosolic protein fraction (which may contain upstream signaling
molecules such as ARFs) to membranes containing GLUT-4 resulted in a transfer of
GLUT-4 proteins from large to small membrane structures. This transfer could be
4
inhibited by neomycin (a PLD inhibitor). Nevertheless, we were not able to replace the
unknown cytosolic molecule(s) by adding recombinant myristoylated ARF1 or ARF6.
Neither did an ARF1 inhibitory peptide block the GLUT-4 transfer. Taken together, the
previous data (17) suggested that PLD could indeed be involved in the GLUT-4
mobilization process, but it was probably not an ARF sensitive PLD1. We have now
taken advantage of the in vitro assay and by protein fractionation we have purified a
protein fraction which induces in vitro GLUT-4 transfer. This fractionation procedure
resulted in co-purification of a PLD which is known to cleave glycosylphosphatidylinositol anchors (7;15).
5
METHODS.
We took advantage of the partial in vitro reconstitution of the GLUT-4 translocation
mechanism (17). In brief, an enriched GLUT-4 membrane fraction (approximately 120 µg of
protein) was isolated by density gradient centrifugation of a post nuclear supernatant. This
enriched GLUT-4 fraction was added control buffer or a cytosolic protein fraction
(approximately 225 µg of protein) (see below). Small and large membrane structures were
then separated by differential centrifugation. The GLUT-4 protein content was determined in
the supernatant containing the small membrane structures and in the pellet containing larger
membrane structures.
Method for isolation of the unknown cytosolic protein. It was found that the unknown
cytosolic factor was present in large amounts in human blood. Thus, forty ml of plasma
proteins were precipitated with polyethylene glycol (9 % wt/vol) and to the supernatant (10
min, 4000 g, 40C) an equal volume of 250 mM bis tris, pH 6.35 was added. All
chromotographic steps were carried out with a Biologic HR workstation (Bio-Rad, Hercules,
CA) operating at 50C. The purification process was monitored and controlled with a Biologic
HR software system. First, the solution was run (flow 3 ml/min) through a column (2.5 cm x
20 cm) containing Q SepharoseTM fast flow (Amersham Pharmacia Biotech A/B,
Buckinhamshire, UK). Non-captured proteins were washed out of the column with 150 ml of
50 mM bis tris, 10 mM NaCl, pH 6.35. Bound protein was eluted with a linear increase in
NaCl to a final concentration of 500 mM. Active fractions eluted in a broad peak around 30
mS/cm. Solid NaCl was added to a final concentration of 500 mM to the active fractions
6
which were then subjected to immobilized metal affinity chromatography (IMAC). A
column (1.5 cm x 5 cm) prepacked with iminodiacetic acid (Pierce, Rockford, Illinois) was
activated with 500 mM zinc acetate and equilibrated with 50 mM tris, 500 mM NaCl, pH 7.5
(flow 1 ml/min). The sample was run through the column, and washed with equilibration
buffer. Captured proteins were eluted with equilibration buffer containing 10 mM histidine.
Active fractions were then separated by size exclusion chromatography (Sephacryl S-200
HR) on a 2.5 cm x 100 cm column (flow 1 ml/min) using equlibration buffer. The GLUT-4
mobilizing activity eluted in 1-2 fractions. Proteins in the active and neighboring fractions
were then separated by SDS polyacrylamide gel (7.5%) electrophoresis. The gel was silver
stained using a kit (Amersham Pharmacia Biotech A/B, Buckinghamshire, UK) in order to
visualize all proteins.
Isolation of the GLUT-4 protein. Approximately 20 g of fresh unfrozen rat skeletal
muscle was transferred to 100 ml of homogenization buffer (50 mM tris base 25 mM,
NaCl, 0.5 mM phenylmethylsulfonyl flouride, pH 7.4) and blended for 1 min. The
homogenate was centrifuged (10 min, 1.500 g, 40C) and the resulting supernatant was
centrifuged (30 min, 230.000 g, 40C). The membrane pellet was resuspended in 30 ml of
ice cold strip buffer (100 mM NaCO3 pH 10.0) and repelleted. This pellet was
resuspended in solubilization buffer (50 mM Mes, 50 mM bis tris, 25 mM NaCl, 0.20 %
(vol/vol) triton X-114, pH 5.5) to a final protein concentration of 0.20 mg/ml. The
samples were left on ice for 30 min with agitation. The samples were centrifuged (30
min, 230.000 g, 40C) followed by filtration of the supernatant through a 0.25 µm syringe
driven filter (Millipore). The supernatant was run (flow 3.0 ml/min) through a
7
chromatography column (2.5 cm x 20 cm) containing Q SepharoseTM fast flow
(Amersham Pharmacia Biotech AB, Uppsala, Sweden) equilibrated in solubilization
buffer. The column was washed with 100 ml of solubilization buffer. The GLUT-4
protein was eluted with a linear increase in the NaCl concentration (25 mM – 1 M) in
solubilization buffer. Fractions containing GLUT-4 were dissolved in a equal volume of
5 mM sodium phosphate buffer pH 6.8 containing 0.2 % (vol/vol) triton X-114 and
subjected to a column containing ceramic hydroxyapatite (Bio-Rad, Hercules, CA).
Bound GLUT-4 was eluted with a linear increase in sodium phosphate (5-200 mM)
containing 0.2% triton X-114. Fractions containing GLUT-4 were captured on a Mono Q
(5/5) column (Amersham Pharmacia Biotech) equilibrated with 50 mM tris, 10 mM
NaCl, 0.2 % (vol/vol) triton X-114, pH 7.4. The GLUT-4 protein was eluted by a linear
increase in the NaCl concentration (10 to 500 mM). Based on silver staining, the fractions
contained exclusively the GLUT-4 protein. If the GLUT-4 protein was prepared for [125I]
3-(trifluromethyl)-3-(m-[125I]iodophenyl) diazirine (TID) photo affinity labeling,
detergent was removed from the Mono Q column by extensively washing with sodium
phosphate buffer, pH 8.5 before the GLUT-4 protein was eluted with an linear increase
(0-1 M) in NaCl dissolved in sodium phosphate buffer.
Photo affinity labeling of GLUT-4. The GLUT-4 was upconcentrated by centrifugation
through an amicon 10 filter (Amicon). Ten µCi 3-(trifluromethyl)-3-(m-[125I]iodophenyl)
diazirine (TID) (Amersham Pharmacia Biotech, Buckingham-shire, UK) were added to
the sample which was photo affinity labeled for 30 min at 350 nm. (26;29). For the
detection of phospholipids, the GLUT-4 sample was loaded on a chomotography column
8
(1.5 cm x 5.0 cm) containing Q SepharoseTM fast flow equilibrated in 5 mM sodium
phosphate pH 8.5. The labeled GLUT-4 was loaded on the column, and most of the
unlabelled 125I TID was washed out of the column by 50 ml of buffer and at a flow of 1
ml/min operated with a P1 pump device (Amersham Pharmacia Biotech, Buckinghamshire, UK). Fractions of 1 ml were collected by elution with 5 mM sodium phosphate pH
8.5 containing 1 M NaCl. Five µl 6 M HCl and 200 mM sodium nitrite were added to all
fractions which were subsequently incubated at 600C for 4 hours. Positive GLUT-4 as
well as negative control fractions were added 500 µl of chloroform: methanol (2:1),
followed by centrifugation. The chloroform phase was collected and thin-layer
chromatography was performed on 20 x 20 cm silica gel plates (Sigma) developed in a
solvent of chloroform: methanol: water (65: 25: 4). For the detection of neutral lipids, the
photoaffinity labeled GLUT-4 was dried under a stream of nitrogen to remove the
volative unlabeled 125[I] TIDE. Two hundred µl 0.1 M NaOH were added to the sample
which was then incubated overnight at 40C, and then added 500 µl chloroform: methanol
(2:1) and acetic acid to neutralize the NaOH. The chloroform phase was loaded on a TLC
plate and developed in solvent of hexane:diethylether:acetic acid (60:30:1). The
developed plate was dried and radioactive components were detected with a STORM
phosphoimager (Molecular Dynamics). The positions of phospholipid standards were
detected by submerging the TLC plate in 10% CuSO4 8% phosphoric acid, and baking
the plate for 1h at 1000C. The palmitic acid standard was detected by exposure of dried
plates to iodine vapor.
9
SDS polyacrylamide gel electrophoresis and Western blotting. Proteins were separated on
SDS polyacrylamide gels. Some gels were silver stained using a commercial kit
(Amersham Pharmacia Biotech A/B) or proteins were electrotransferred to PVDF
membranes. The PVDF membranes were Ponceaus S stained, destained and blocked with
3% (wt/vol) bovine serum albumin in TS buffer (50 mM tris, 150 mM NaCl, 0.05%
(vol/vol) NP40, 0.05 % (vol/vol) Tween-20, pH 7.4) for 1.5 hour at room temperature.
The PVDF membranes were then incubated with a primary antibody dissolved in TS
buffer containing 1 % (wt/vol) bovine serum albumin and 0.02% NaN3 (the antibodies
were a goat anti GLUT-4 IgG, a rabbit anti-cross reacting determinant (CRD) IgG, or a
mouse monoclonal anti GPI-PLD IgG purchased from Santa Cruz Biotechnology, Santa
Cruz, CA., Glyko, Inc., Novato, CA., or Transduction Laboratories, Lexington, KY.,
respectively). Antigen and primary antibody complexes were visualized upon binding
with a secondary antibody conjugated with alkaline phosphatase (DAKO, Glostrup,
Copenhagen, Denmark) and an enhanced chemofluorescense kit (Amersham Pharmacia
Biotech A/B). Fluoroscense were detected with a STORMTM phosphoimager (Molecular
Dynamics).
Statistics. All results are shown as mean values ± standard error (SE) with a significance
level at p < 0.05. Paired and un-paired t-tests were used to calculate the p level.
10
RESULTS
In this paper we have used a previously developed assay which displays in vitro GLUT-4
transfer from large to small membrane structures (17). In brief, the assay is performed by
incubation of a mixture of an enriched GLUT-4 membrane fraction and cytosolic
proteins. The GLUT-4 protein content is then determined in the fast pelleting large
membrane fraction and in the slow pelleting small membrane fraction, as shown in figure
1, upper panel. In particular, the increase in the GLUT-4 protein content in the small
membrane fraction is not caused by adding GLUT-4 protein contained in the cytosol,
since no GLUT-4 signal could be detected in the supernatant and pellet in experiments in
which cytosol was added alone.
The in vitro GLUT-4 assay is dependent upon the prior in vivo physiological condition of
the rat. In brief, by adding unfrozen insulin-stimulated (1 min) cytosol, basal or no
cytosol, respectively, there is an increase in GLUT-4 transfer from large to small
membrane structures in an enriched GLUT-4 fraction isolated from a basal skeletal
muscle (figure 1, middle panel). However, unfrozen cytosol from insulin-stimulated
unfrozen skeletal muscle was only approximately 10-20% more potent when compared to
unfrozen basal cytosol. This order of potency between basal and insulin stimulated
cytosol is diminished with frozen cytosol (data not shown). In particular, adding basal or
insulin-stimulated (1 min) cytosol to an enriched GLUT-4 membrane fraction from prior
insulin-stimulated (10 min) skeletal muscle was not as potent as using basal skeletal
11
muscle as a source of the enriched GLUT-4 donor membrane fraction. We tested whether
cytosolic protein fractions prepared from other tissue sources (liver, brain, blood plasma)
than rat skeletal muscle could induce in vitro GLUT-4 transfer. We found that human
blood plasma and to a lesser extent brain very potently induced in vitro GLUT-4 transfer,
as shown in figure 1, lower panel. Thus, human blood plasma seemed to be a good source
of the unknown stimulating factor and we therefore set out to identify the unknown
stimulating factor found in the plasma. By protein chromatography of plasma we
identified elute fractions, which were able to induce in vitro GLUT-4 transfer. First, to
the resulting supernatant from polyethylen glykol precipitation, 50 mM of mes, bis-tris,
or tris buffer was added to give different pH values (5.8, 6.3 and 7.8). The solutions were
subjected to anion as well as cation exchange. In brief, it was found that the unknown
factor did bind to a cation resin at pH 5.8 but not at 6.3 and 7.8. The factor did also bind
to anion resin at pH 7.8 and at 6.3 with a lower affinity, but not at 5.8 (data not shown).
This suggested that the isoelectric point for the protein(s) was between 5.8-6.3. We then
performed anion exchange at pH 6.35. The procedure for identification of the unknown
protein is outlined in figure 2. Elute fractions able to induce GLUT-4 transfer in the in
vitro assay where then collected. Since we have previously observed that the in vitro
GLUT-4 transfer is sensitive to the concentration of divalent cations, we performed metal
affinity chromatography. Indeed, the fractions which eluted with the chelating amino acid
histidine were able to induce GLUT-4 transfer. These fractions were then protein
fractionated using a third parameter namely size exclusion chromatography. Interestingly,
only 1-2 elute fraction(s) were able to induce GLUT-4 transfer. The elution profile
suggested that the native protein had a molecular size higher than the range of 200-250
12
kDa. The active 1-2 fraction(s) and the neighboring non-active fractions were
electrophoresed on 7.5 % gel, as shown in figure 2. All proteins were detected by silver
staining in order to detect the presence of an extra band in the active fractions when
compared to the non-active fractions. Only one extra band (around 110 kDa) appeared in
the active fractions and these extra bands were identified by immunoblotting to be GPI
PLD (figure 2c).
Based on our observations regarding the source for purification (plasma), iso-electric
point (5.8-6.3), presence of histidine in the protein, and the molecular weight of the
native and denatured protein (200-250 kDa and 110 kDa), and our previous observation
of production of phosphatidic acid in GLUT-4 immunoprecipitates, we hypothesized that
the unknown protein could be GPI-PLD (20;30). This was tested with a GPI-PLD
specific antibody. Indeed, GPI-PLD eluted exclusively in the active fraction(s) able to
induce GLUT-4 transfer and the immunblot signal appeared at the same molecular weight
as the extra band in the silver stain (data not shown).
This suggested that the GLUT-4 containing vesicles could be linked to a large membrane
compartment via a GPI link (see figure 9). We wanted to verify this hypothesis. First, we
added 10 mM of suramin and 1 mM of 1,10 phenanthroline (an activator (21) and
inhibitor (24) of membrane bound GPI PLD activity, respectively) to the GLUT-4 in vitro
assay. As shown in figure 3, upper panel, suramin was able to potentiate the effect of
plasma. Interestingly, suramin did also pontentiate the GLUT-4 transfer in the absence of
plasma proteins, suggesting that some of the GPI-PLD could be associated with the large
13
membrane compartment. Finally, 1 mM of 1,10 phenanthroline did inhibit the in vitro
GLUT-4 transfer, figure 3, lower panel.
Furthermore, we wanted to purify the GLUT-4 protein in order to chemically verify the
co-purification of a GPI link. The procedure for purifying the GLUT-4 protein is outlined
in figure 4. In brief, the GLUT-4 protein was isolated from a rat skeletal muscle
homogenate. Next, a membrane preparation was obtained by centrifugation and
peripheral membrane proteins were stripped off. The integral membrane proteins were
solubilized and purified by anion-exchange and hydroxyapatite chromatography. The
GLUT-4 transporter was more than 99% pure based on silver staining of proteins in a
10% SDS gel (figure 4, upper panel). Interestingly, the GLUT-4 protein did bind to the
positive anion resin at pH 5.5 which is a much lower pH value than the calculated
isoelectric point based on the polypeptide sequence. Thus, an extra negative charge must
be bound directly to the GLUT-4 protein. In fact, when the elute fraction from the final
chromatography Mono Q step was separated on a gel and silver stained the GLUT-4
protein eluted in 3 bands around 50 kDa. All 3 bands were recognized by the anti-GLUT4
IgG. The triplets were not uniformly distributed in the elute fractions since the high
molecular GLUT-4 eluted before the low molecular GLUT-4 isotype. The high molecular
isotype may then have a higher isoelectric point (carrying relative more positive charges
at the same pH) which causes elution prior to the relative more negative low molecular
isoform. This could be explained by the presence (non-cleaved) or absence (cleaved) of a
negatively charged phosphate molecule as part of a GPI link (see figure 9 in discussion).
Furthermore, it may also explain why the GLUT-4 protein could bind to the positive
14
anion resin at a lower pH value than predicted from the calculated isoelectric point. The
calculated isoelectric point is exclusively based on the polypeptide sequence and does not
include conjugated site-groups.
Next, we wanted to see whether we could recover constituents from a GPI link by
labeling of all reactive site groups found on the isolated GLUT-4 protein. In brief, the
GLUT-4 protein was captured on a chromatography column that was extensively washed
with detergent in order to remove all lipids. The GLUT-4 protein was then eluted without
detergent and the purified GLUT-4 transporter and its reactive chemical site groups were
photo affinity labeled with [125I] TID. In experiments designed for detection of
phospholipids, the labeled GLUT-4 protein was again captured on an anion-exchange
column, and the excess of free unlabelled [125I] TID was washed away. The GLUT-4
protein was then eluted from the column and all elute fractions were nitrous deaminated.
Fractions containing GLUT-4 were then identified. Lipids were then extracted from
fractions with GLUT-4 and from fractions devoid of GLUT-4 and separated on a thin
layer chromatography plate, as shown in figure 6. A clear radioactive signal appeared in
the positive GLUT-4 fractions. No signal was detected in labeled control fractions
devoid of GLUT-4 suggesting that the signal originated from the GLUT-4 protein. The
signal had a migration pattern similar to phosphatidylethanolamine, an known constituent
in a GPI link. Furthermore, for the detection of fatty acids the photo affinity labeled
GLUT-4 was hydrolysed before any possible lipids were extracted and separated on a
thin layer chromatography plate. Interestingly, now 2 radioactive bands appeared with a
migration pattern similar to the palmitic acid standard.
15
Furthermore, we took advantage of the isolation of GLUT-4 protein in the presence of the
detergent triton X-114. Logically, the GLUT-4 protein was detected in a Western blot in
the water-detergent solution, as shown in figure 7, upper panel. By phase transition and
separation of water from pure detergent, proteins could be separated into the detergent
phase and water phase. The GLUT-4 could be immunodetected in the detergent phase,
but hardly any signal was detected from the water phase. It should be noted that
separation of detergent from the water phase results in a large volume of water and a
minor volume of detergent. However, only a fraction of the water volume is run on the
gel due to the size of the well in the gel. Thus, the GLUT-4 content in the water phase is
higher than indicated in the figure. When the protein was upconcentrated by ammonium
sulfate precipitation, the protein in the water phase did in fact react with a GLUT-4
specific antibody as shown in figure 7, lower panel. This strongly suggest the presence of
GLUT-4 in the water phase. This is a known behaviour for GPI linked proteins.
Finally, we separated the pure GLUT-4 transporter on a SDS gel and immunoblotted with
an CRD antibody raised against an epitope which is uniquely formed on GPI proteins
treated with phosphatidylinositol PLC (4;16). The CRD antibody reacted against a
positive standard prepared from the variant surface glycoprotein prepared from T. Brucei.
Interestingly, this antibody did also cross-react with the GLUT-4 transporter, as shown in
figure 8.
16
DISCUSSION.
We here report on the isolation of a GPI PLD, which may be able to release GLUT-4
containing vesicles from membranes by cleavage of a GPI link on the GLUT-4
transporter. Furthermore, we purified the GLUT-4 protein and provide immunological as
well as biochemical evidence for the novel concept that GLUT-4 may indeed contain
such a GPI link (figure 9).
We have previously published evidence that a PLD may be involved in the mobilization
of GLUT-4 containing vesicles in skeletal muscle (17). This was primarily based on the
finding that in vivo insulin stimulation resulted in an increased production of
phosphatidic acid in the GLUT-4 immunoprecipitate isolated from a low spin membrane
fraction, but not in GLUT-4 immunoprecipitate isolated from a high spin membrane
fraction. In the present study we have taken advantage of this in vitro assay and we have
purified a protein fraction which induces GLUT-4 transfer in our assay. This fraction
contained a PLD which is known to cleave glycosylphosphatidylinositol (GPI) anchors
(7;15). The cleavage of GPI-linked proteins by GPI-PLD results in production of
phosphatidic acid in the large membrane compartment harboring part of the GPI link (see
figure 9). This may explain our previous finding of an insulin-induced production of
phosphatidic acid in the GLUT-4 membrane compartment immunoprecipitated from a
low spin membrane fraction (17). By adding PLD inhibitor/activators to the in vitro assay
and by isolation of the pure GLUT-4 transporter we provide evidence that the GLUT-4
17
containing vesicles may be GPI linked to a large donor membrane compartment. Upon
stimulation this GPI link may be cleaved which may release the GLUT-4 containing
vesicles from binding to membranes.
It should be noted that we used plasma as the source of purifying the GPI PLD.
Unfortunately, we could not immunologically test whether GPI PLD is found in skeletal
muscle, since there are no antibodies against rodent GPI PLD. Furthermore, due to the
contamination of human muscle biopsies with plasma it is not possible to verify whether
the GPI PLD originates from plasma or the muscle cell itself. However, GPI PLD mRNA
expression is observed in skeletal muscle (20). Furthermore, the production of
phosphatidic acid in GLUT4 immunoprecipitates (17), the co-isolation of the GPI
constituents phosphatidylethanolamine and fatty acids with the pure GLUT-4 transporter
(figure 6), and the effect of suramin and 1,10 phenanthroline on the in vitro GLUT-4
transfer carried out with cytosol isolated from rat skeletal muscle (figure 3), very strongly
indicate the enzymatic involvement of a GPI PLD in skeletal muscle.
Interestingly, there was no major difference between the potency of using cytosol from
basal skeletal muscle when compared to insulin-stimulated (1 min) cytosol. This may
suggest a constant active GPI PLD in the prepared cytosol. However, this is probably an
artifact caused by homogenization and may not necessarily be a reflection of the in vivo
GPI PLD activity in the intact muscle cell. For instance, the in vivo GPI PLD activity is
related to the concentration of divalent cations (7;8;15;22;24). In fact, chelating the
divalent cations by adding 1,10 phenanthroline in the in vitro GLUT-4 assay very
18
potently inhibited the GLUT-4 transfer (figure 3). Furthermore, GLUT-4 transfer was
sensitive to the magnesium concentration (17). Although the concentrations of cations in
the buffer are made comparable to the intracellular cytosolic concentrations, the true
intracellular concentrations may differ due to membrane compartmentalization. Another
alternative is that the GPI PLD in fact is constantly active in vivo in intact cells. If so the
release of GLUT-4 containing vesicles may be clamped by another mechanism. We and
others have observed docking to and phosphorylation of GLUT-4 protein by PKBβ in the
process of GLUT-4 mobilization from membranes (3;17;19). This suggest to us that
PKBβ (or possibly another protein kinase) may phosphorylate the GLUT-4 protein
preparing it for GPI PLD cleavage. If GLUT-4 phosphorylation is a prerequisite for PLD
cleavage the docking of PKBβ could be the regulated step in the GLUT-4 mobilization
process.
The GPI PLD also shows sequence similarity with the integrin family (15). This family is
known for mechanochemical transduction properties via PI-3 kinase, which may suggest
that GPI PLD could be activated by both insulin and muscle contractions which are the
two physiological stimuli of GLUT-4 translocation to the surface membrane of muscle
cells. In this regard, it should be noted that the production of phosphatidic acid in the
GLUT-4 immunoprecipitate was insulin-sensitive (17) and furthermore that muscle
contractions cause production of phoshatidic acid in rat skeletal muscle in vivo
suggesting activation of a PLD (6). Interestingly, when GPI is added to cells it mimics
many metabolic actions of insulin (25) suggesting that the GPI link has signaling
properties of its own (2;13).
19
It was observed that GLUT-4 had an elution profile where charge was related to a change
in molecular weight. It was speculated that this finding could be due to the presence
(uncleaved) and absence (cleaved) of a phosphate as part of the GPI link (see figure 5).
Given this, we were also curious to examine the elution profile for GLUT-4 isolated from
basal and insulin stimulated skeletal muscle in order to try to detect a change in
molecular weight and elution profile. Unfortunately, the poor linearity with silver
staining and protein content and the strong signal in the Western blot made it difficult to
make any measurements regarding small changes in molecular weight/charge and its
possible relation to stimulation with insulin. It was also observed that the GLUT-4
protein could be separated into 2 different phases, namely a water phase and a detergent
phase. This is at first glance very unexpected for an integral membrane protein. However,
it is a very well known observation for GPI linked proteins (4;12;26), since the link is
detergent resistant (11) and contains large and bulky side chains consisting of very water
soluble glucosamine, carbohydrates and phosphate. Thus, the present observations
strengthen the concept that GLUT-4 protein contains a GPI link. Finally, recent reports
suggest that insulin-stimulated GLUT-4 translocation requires the translocation of cbl to
lipid rafts (5) which are detergent resistant membrane areas enriched in GPI linked
proteins (27).
One question is whether all GLUT-4 transporters inside the muscle cell are released via
this suggested mechanism. We have used an in vitro assay which may - or may not represent the whole population of GLUT-4. We have previously done marker enzyme
20
analysis on this membrane fraction (17). However, prior membrane homogenization
causes cross contamination of membranes and enzyme marker analysis on the recovered
membrane fraction does not discriminate between the GLUT-4 positive membrane and
membranes devoid of GLUT-4. Thus, we are not in a position to answer this question
with the techniques used in the present study. Future studies using microscopy of
immunolabelled membrane organelles are necessary to answer this question.
A number of consensus polypeptide sequences are known to signal for the attachment of
a GPI (31). This attachment is mediated by a transamidase reaction and takes place
during synthesis of the polypeptide in the endoplasmatic reticulum. By using computer
based prediction analysis, it was suggested that the C-terminal of GLUT-4 (the amino
acid # 480, SAT sequence) potentially could have a GPI anchor sequence. However, this
prediction is based on calculation following the ω,ω+2 rule and may produce false
positive results (31). The family of GPI linked proteins is broad (10) and many GPI
linked proteins are localized on the exterior of the cells, where they can be released into
the surrounding milieu. Given this, it might seem surprising that the intracellular located
GLUT-4 protein should contain a GPI link. However, also ornithine decarboxylase, GPI
proteins in granules and trafficking of GPI are related to the interior of the cell (1;10;12).
Furthermore, some aminopeptidases are also known to be GPI linked and released by this
mechanism (23). Interestingly, one of the constituents in the GLUT4 containing vesicles
is IRAP/-aminopeptidase N. This is one of the few proteins found in the GLUT-4
immuno-precipitate which fully translocates together with GLUT-4 to the surface
membrane. It is tempting to speculate that GLUT-4 and IRAP/aminopeptidase are
21
sequestered in the same vesicle because they are both released by cleavage of a GPI link.
If IRAP and GLUT-4 indeed are sequestered in the same translocating vesicle it might be
argued that in vitro GLUT-4 transfer (as observed when adding GPI PLD to the GLUT-4
donor membranes in the present study) may be due to cleavage of a GPI link to the IRAP
rather than to GLUT-4. However, our finding of a GPI link on the pure GLUT-4 protein
indicates that the GPI link indeed is on GLUT-4.
Taken together, the present paper offers several novel findings. First, the further
characterization and description of an assay strategy for studying ongoing enzymatic
reactions in a demanding tissue. Second, a method was developed for purifying large and
pure amounts of GLUT-4 proteins enabling GLUT-4 immobilization and studies of
GLUT-4 phosphorylation and binding by other proteins. Third, we have shown that GPI
PLD may cause the release of GLUT-4 containing vesicles from an enriched GLUT-4
fraction. This GLUT-4 release could be activated and inhibited with suramin and 1,10
phenanthroline, compounds known to activate and inhibit GPI PLD activity, respectively.
By purifying the GLUT-4 transporter we also provide immunological and biochemical
evidence for a GPI link. In conclusion, we suggest that a GPI PLD may be a novel
important mediator for GLUT-4 mobilization in skeletal muscle.
22
Acknowledgements:
The present study was supported by grants from the Danish National Research
Foundation (504-14) and from Novo-Nordisk Research Foundation and Danish Diabetes
Association.
23
Reference List
1. Bauman, N. A., J. Vidugiriene, C. E. Machamer, and A. Menon. Cell surface
display and intracellular trafficking of free glycophosphatidylinositols in
mammalian cells. J. Biol. Chem. 275: 7378-7389, 2001.
2. Bruni, P., E. Meacci, M. Avila, V. Vasta, M. Farnararo, J. M. Mato, and I.
Varela. A phospho-oligosaccharide can reproduce the stimulatory effect of insulin
on glycolytic flux in human fibroblasts. Biochem. Biophys. Res.Comm. 166: 765771, 1990.
3. Calera, M. R., C. Martinez, H. Liu, A. E. J. Jack, M. Birnbaum, and P. F.
Pilch. Insulin increases the association of Akt-2 with GLUT4-containing vesicles.
J. Biol. Chem. 273: 7201-7204, 1998.
4. Cardoso de Almeida, M. L. and M. J. Turner. The membrane form of variant
surface glycoproteins of Trypanosoma brucei. Nature 302: 349-352, 1983.
5. Chiang, S., C. Bauman, M. Kanzaki, D. Thurmond, R. Watson, C. Neudauer,
I. Macara, J. Pessin, and A. Saltiel. Insulin-stimulated GLUT4 translocation
requires the CAP-dependent activation of TC10. Nature 410: 944-948, 2001.
24
6. Cleland, P. J., G. Appleby, S. Rattigan, and M. Clark. Exercise-induced
translocation of protein kinase C and production of diacylglycerol and phosphatidic
acid in rat skeletal muscle in vivo. J. Biol. Chem. 264: 17704-17711, 1989.
7. Davitz, M. A., J. Hom, and S. Schenkman. Purification of a glycosylphosphatridylinositol-specific phospholipase D from human plasma. J. Biol. Chem.
264: 13760-13764, 1989.
8. de Almeida, M. L., M. J. Turner, B. B. Stambuk, and S. Schenkman.
Identification of an acid-lipase in human serum which is capable of solubilizing
glycophosphatidylinositol-anchored proteins. Biochem. Biophys. Res. Comm. 150:
476-482, 1988.
9. Emoto, M., J. K. Klarlund, S. B. Waters, V. Hu, J. M. Buxton, A. Chawla, and
M. P. Czech. A role for phospholipase D in GLUT4 glucose transporter
translocation . J. Biol. Chem. 275: 7144-7151, 2000.
10. Ferguson, M. A. and A. F. Williams. Cell-surface anchoring of proteins via
glycosyl-phosphatidylinositol structures. Ann. Rev. Biochem. 57: 285-320, 1988.
11. Fiedler, K., T. Kobayashi, T. V. Kurzchalia, and K. Simons. Glycosphingolipidenriched, detergent-insoluble complexes in protein sorting in epithelial cells.
Biochemistry 32: 6365-6373, 1993.
25
12. Fouchier, F., P. Bastiani, T. Baltz, D. Aunis, and G. Rougon.
Glycosylphosphatidylinositol is involved in the membrane attachment of proteins in
granules of chromaffin cells. Biochem. J. 256: 103-108, 1988.
13. Frick, W., A. Bauer, J. Bauer, S. Wied, and G. Muller. Insulin-mimetic
signalling of synthetic phosphoinositolglycans in isolated rat adipocytes. Biochem
J. 336 ( Pt 1): 163-181, 1998.
14. Hammond, S. M., J. M. Jenco, S. Nakashima, K. Cadwallader, Q.-M. Gu, S.
Cook, Y. Nozawa, G. D. Prestwich, M. A. Frohman, and A. J. Morris.
Characterization of two alternatively spliced forms of phospholipase D1. J. Biol.
Chem. 272: 3860-3868, 1997.
15. Huang, K. S., S. Li, W. J. Fung, J. D. Hulmes, L. Reik, Y. C. Pan, and M. G.
Low. Purification and characterization of glycosyl-phosphatidylinositol- specific
phospholipase D. J. Biol. Chem. 265: 17738-17745, 1990.
16. Jones, D. R., M. A. Avila, C. Sanz, and I. Varela-Nieto. Glycosylphosphatidylinositol-phospholipase type D: a possible candidate for the generation
of second messengers. Biochem. Biophys. Res. Comm. 233: 432-437, 1997.
17. Kristiansen, S., Nielsen, JN., Bourgoin, S., Klip, A., Franco, M., and Richter,
E. A. GLUT-4 translocation in skeletal muscle studied with a cell-free assay:
involvement of phospholipase D. Am. J. Physiol. 281: E608-E618, 2001.
26
18. Kristiansen, S., T. Ramlal, and A. Klip. Phosphatidylinositol 4-kinase, but not
phosphatidylinositol 3-kinase, is present in GLUT4-containing vesicles isolated
from rat skeletal muscle. Biochem. J. 335 ( Pt 2): 351-356, 1998.
19. Kupriyanova, T. A. and K. V. Kandror. Akt-2 binds to Glut4-containing vesicles
and phosphorylates their component proteins in response to insulin. J. Biol. Chem.
274: 1458-1464, 1999.
20. LeBoeuf, R. C., M. Caldwell, Y. Guo, C. Metz, M. A. Davitz, L. K. Olson, and
M. A. Deeg. Mouse glycosylphosphatidylinositol-specific phospholipase D (Gpld1)
characterization. Mamm.Genome 9: 710-714, 1998.
21. Lee, J. Y., M. R. Kim, and D. E. Sok. Enzymatic release of Zn2+glycerophosphocholine cholinephosphodiesterase from brain membranes by
glycosylphosphatidylinositol-specific phospholipases and its regulation.
Neurochem. Res. 23: 899-905, 1998.
22. Li, J. Y., K. Hollfelder, K. S. Huang, and M. G. Low. Structural features of GPIspecific phospholipase D revealed by proteolytic fragmentation and Ca2+ binding
studies. J. Biol. Chem. 269: 28963-28971, 1994.
23. Low, M. G. The glycosyl-phosphatidylinositol anchor of membrane proteins.
Biochim. Biophys. Acta 988: 427-454, 1989.
27
24. Metz, C. N., G. Brunner, N. H. Choi-Muira, H. Nguyen, J. Gabrilove, I. W.
Caras, N. Altszuler, D. B. Rifkin, E. L. Wilson, and M. A. Davitz. Release of
GPI-anchored membrane proteins by a cell-associated GPI- specific phospholipase
D. EMBO J. 13: 1741-1751, 1994.
25. Misek, D. E. and A. R. Saltiel. An inositol phosphate glycan derived from a
Trypanosoma brucei glycosyl- phosphatidylinositol mimics some of the metabolic
actions of insulin. J. Biol. Chem. 267: 16266-16273, 1992.
26. N.M.Hooper. Identification of a glycosylphosphatidylinositol anchor on membrane
proteins. In N.M.Hooper and A.J.Turner. Lipid modifications of proteins: A
practical approach. Leeds, IRL press. 1992, 89-113.
27. Pfeffer, S. Caveolae on the move. Nature Cell Biology 3: E108-E110, 2001.
28. Ploug, T., B. Deurs, H. Ai, S. W. Cushman, and E. Ralston. Analysis of GLUT4
distribution in whole skeletal muscle fibers: Identification of distinct compartments
that are recruited by insulin and muscle contractions. J. Cell Biol. 135: 415-430,
1998.
29. Roberts, W. L., B. H. Kim, and T. L. Rosenberry. Differences in the glycolipid
membrane anchors of bovine and human erythrocyte acetylcholinesterases. Proc.
Natl. Acad. Sci.U.S.A 84: 7817-7821, 1987.
28
30. Stadelmann, B., A. Zurbriggen, and U. Brodbeck. Distribution of
glycosylphosphatidylinositol-specific phospholipase D mRNA in bovine tissue
sections. Cell Tissue Res. 274: 547-552, 1993.
31.
Udenfriend, U. and K. Kadukula. Prediction of site in nascent precusors of
glycosylphosphatidylinositol protein. Casey, P. J. and J. E. Buss. Methods in
Enzymology. San Diego, Academic Press. 1995, 571-581.
29
Legend 1.
Upper panel. An enriched GLUT-4 membrane fraction was isolated from basal (B) rat
skeletal muscle and incubated for 45 min at 370C with no cytosol (none), or a cytosolic
protein fraction isolated from basal (B) skeletal muscle. The large and small membrane
structures were subsequently separated by a differential centrifugation step. Proteins from
the 2 fractions were subjected to GLUT-4 Western blotting. The upper panel shows one
representative immunoblot (double determination) out of approximately 50.
Middle panel: An enriched GLUT-4 membrane fraction was isolated from basal (B) rat
skeletal muscle or insulin (Ins)-stimulated (10 min) rat skeletal muscle and incubated
with no cytosol (none), or a cytosolic protein fraction isolated from basal (B) or insulin
(Ins)-stimulated (1 min) rat skeletal muscle. The middle panel shows a representative
GLUT-4 Western blot of 4 experiments, each done with double determination of each
experimental condition.
Bottom panel. An enriched GLUT-4 membrane fraction was isolated from basal skeletal
muscle and incubated with no cytosol (none), cytosol prepared from rat skeletal muscle
(Sm), rat liver (Li), rat brain (Br) and rat blood plasma (Bp). The immunoblot is
representative of approximately 50, 2, 2, 28 experiments for skeletal muscle, rat liver, rat
brain, and blood plasma, respectively.
30
Legend 2.
The unknown protein factor was identified by testing different elute fractions for its
capability to induce in vitro GLUT-4 transfer. The upper panel shows the increase in
GLUT-4 content in the small vesicle fraction as a result of adding a control non-active
fraction (lane 1), the active protein fraction from Q sepharose (lane 2), the active elute
fraction from Zn++ immobilized metal chromatography (lane 3) and adding fraction # 39
and 40 from the size exclusion chromatography experiment (lane 4). The fractions
ranging from 36 to 42 from the size exclusion chromatograhy step were examined closer
by gel electrophoresis and silver staining as indicated in the middle panel. The active
fractions # 39 and # 40 contained a distinct protein band at 110 kDa (marked with an
arrow). The protein fractions were also electro transferred to a membrane and probed
with an anti GPI PLD IgG as indicated in the lower panel. The elute fractions and stained
gel is representative of 4 different experiments.
31
Legend 3.
Upper panel: The upper panel shows the effect of 10 mM of suramin (a GPI PLD
activator) on the in vitro GLUT-4 transfer . An enriched GLUT-4 membrane fraction was
isolated from basal rat skeletal muscle and added nothing (-) or cytosol (+) prepared from
basal skeletal muscle in the presence (+) or absence (-) of suramin. The mixtures were
subsequently differential centrifugated. The graph shows the GLUT-4 protein content in
the supernatant containing the small membrane structures. The calculated mean + SE are
from 4 different experiments, each determination done in duplicate. The bars are no
cytosol (empty bar), cytosol (filled bar), cytosol + suramin (cross hatched bar) and no
cytosol + suramin (hatched bar). * p<0.05 when compared to no cytosol (empty bar). $ p
< 0.05 compared to cytosol (filled bars).
Lower panel. The lower panel shows the effect of 1 mM of 1,10 phenanthroline (an
inhibitor of the GPI PLD activity). As explained in the text to the upper panel, the
enriched GLUT-4 membrane fractions were incubated with or without cytosol and 1,10
phenanthroline, as indicated. The graph shows the calculated mean + SE from 4 different
experiments, each in duplicate. The bars are no cytosol (empty bar), cytosol (filled bar),
cytosol + 1,10 phenanthroline (cross-hatched bar). * p<0.05 when compared to no cytosol
(empty bar).
32
Legend 4.
The figure outlines the purification protocol for isolating the GLUT-4 transporter from rat
skeletal muscle. The upper panel shows a silver stain of all proteins in a 10% gel loaded
with a muscle homogenate (lane 1), a fraction containing peripheral and integral
membrane proteins (lane 2), integral proteins (lane 3), positive GLUT-4 elute fractions
from Q Sepharose (lane 4), hydroxyapatite (lane 5) and Mono Q chromatography (lane 6)
and a molecular weight marker (lane 7). No other proteins than the immunoreactive
GLUT-4 (lower panel) could be observed in lane 6. The silver stain and GLUT-4
immunoblot are representative of 10-15 determinations.
Legend 5.
The figure shows the change in GLUT-4 migration pattern on the gel with increasing
charge loaded on the Mono Q (5/5) column. The silver stain is representative for 4
different elution experiments.
33
Legend 6.
Pure GLUT-4 protein was photo affinity labelled with [125I] TID. In some experiments
the labelled GLUT-4 protein was captured on a Q Sepharose column, washed and eluted
with an increase in sodium chloride. Positive (+) and negative (-) GLUT-4 elute fractions
were subjected to nitrous deamination. Alternatively, unlabelled [125I] TID were removed
from GLUT-4 by drying the sample under a stream of N2. The residual sample was base
hydrolysed. Lipids were extracted from the mixture and run on a thin layer
chromatography plate with 2 different solvents for the separation of phospholipids (top
panel : nitrous deamination) or fatty acids (bottom panel : base hydrolysis). Positive
standards for phosphatidylserine (PS), phosphatidylcholine (PC), phosphatidylinositol
(PI), and phosphatidylethanolamine (Peth.) and palmitic acid (PA) were separated and
stained in the left top panel. An arrow is used to indicate the migration of palmitic acid
standard in the solvent used for base hydrolysis (lower panel). The radioactive signals
shown are representative of 3 different experiments.
34
Legend 7.
The pure GLUT-4 transporter was eluted from a Mono Q (5/5) column in the presence of
the detergent triton X-114. Fractions consisting of the water-detergent (WD) - , the water
(w) or detergent (D) phases were separated on a gel and silver stained or Western blotted
with an anti GLUT-4 IgG. The upper panel is representative of 3 different experiments.
In some experiments, the GLUT-4 protein in the water phase was precipitated and
subjected to GLUT-4 Western blotting (lower panel).
Legend 8.
The pure GLUT-4 transporter was separated on a gel and Western blotted with a cross
reacting determinant (CRD) antibody raised against the GPI link on the variant surface
glycoprotein (VSV) from T Brucei or blotted with an anti-GLUT-4 IgG. The immunoblot
is representative of 4 different experiments.
Legend 9.
The figure shows a hypothetical model of a putative GPI link from the large membrane
structure to the GLUT-4 protein in the vesicle. The link may consist of fatty acids,
phosphate, inositol, glucosamine, several mannitol residues, and phosphatidylethanolamine. The PLD cleavage site is shown.
35
Figure 1
Donor membranes: B
None
B
Large
membrane
structures
Small
membrane
structures
Cytosol :
None
Donor
membranes:
B
B
B
Ins
Small
membrane
structures
Cytosol :
None
B
Ins
B
Ins
Small
membrane
structures
Cytosol:
None
Sm
Li.
Br.
Bp
Figure 2.
36
1
2
3
4
GLUT-4 protein
content in small
membrane structures
<50 kDa
Silver
stain
GPI PLD
Western
blot
36………..………….42
SEC elute fractions
37
Figure 3
GLUT-4
content
in small
membra
ne structures.
$
1.60
$
1.40
1.20
1.00
*
0.80
0.60
0.40
0.20
0.00
Cyt: Suramin: -
GLUT-4
content
in small
membra
ne structures
0.45
+
-
+ + +
*
0.40
0.35
0.30
0.25
0.20
0.15
0.10
0.05
0.00
Cyt: 1,10 ph.thr.: -
+
-
+
+
38
Figure 4
1 2 3 4 5 6
Silver
stain
GLUT-4
Western
blot
7
<50 kDa
< 50 kDa
39
Figure 5
GLUT-4
silver stain
50 kDa >
40
Figure 6
Nitrous deamination
- + - +
PS PC PI PEth PA
Base hydrolysis
PA
+
-
41
Figure 7
WD
W
D
Silver
stain
GLUT-4
Western
blot
<50 kDa
GLUT-4
Western
blot
<50 kDa
D
D
W W
42
Figure 8
VSG
GLUT-4
< 50 kda
CRD IgG
GLUT-4 IgG
43
Figure 9
GLUT-4 containing
vesicle
GLUT-4
polypeptide
Ethanolamine
(Man)3 Glucosamine
C-C-CO O
O=C C=O
P
Inositol
GPI PLD
cleavage site
Large
membrane
structure