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Identification of the glucose transporter in rat skeletal muscle
ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Vol. 226, No. 1, October 1, pp. 198-205, 1983
Identification
of the Glucose Transporter
in Rat Skeletal Muscle’
AMIRA KLIP,2 DENISE WALKER, KATHLEEN J. RANSOME,* DEAN W. SCHROER,* AND GUSTAV E. LIENHARD* Division of Neurology, Research Institute, Canada, and *Department of Biochemistry,
The Hospital for Sick Children, Tunmto, Ontario M5G lx& Dartmouth Medical school, Hanover, New Hampshire OS756
Received March 7, 1983, and in revised form May 27, 1983
The glucose transporter in the plasma membrane of rat skeletal muscle has been identified by two approaches. In one, the transporter was detected as the polypeptide that was differentially labeled by photolysis with [‘Hlcytochalasin B in the presence of L- and D-glucose. [‘HlCytochalasin B is a high-affinity ligand for the transporter that is displaced by D-glucose. In the other, the transporter was detected by means of its reaction with rabbit antibodies against the purified glucose transporter from human erythrocytes. By both procedures, the transporter was found to be a polypeptide with a mobility corresponding to a molecular weight of 45,000-50,000 upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis.
Glucose transport into skeletal muscle cells occurs through a system of the facilitated diffusion type (l-3). This system exhibits several kinds of regulation. Transport is stimulated by the hormones insulin and epinephrine, by contractile activity, and by anoxia (1,2). A recent study has shown that insulin probably enhances glucose transport in muscle by causing the translocation of transporters from an intracellular location to the plasma membrane (4). The bases for the other types of regulation are not known. The actions of epinephrine and contractile activity of muscle differ in part from those of insulin, since the reversal of the stimulation by epinephrine and contractile activity requires protein synthesis, whereas the reversal of the insulin effect does not (5). The glucose-transporter protein in muscle has not been previously identified. Further investigation of these regulatory phenomena would be facilitated by such identification. Recently, two approaches have 1 This paper is the second in a series. No. 1 is Ref. (17). 2 To whom correspondence should be addressed. 0003-9861/83 $3.00 Copyright All rights
0 1983 by Academic Press, Inc. of reproduction in any form reserved.
proven successful in the detection of the glucose transporter in other cell types. In one of these, the transporter was photoaffinity-labeled with [‘Hlcytochalasin B, a high-affinity inhibitor of the transporter. D-Glucose competes with cytochalasin B for reversible binding to the transporter; hence the transporter polypeptide can be detected by its differential incorporation of label in the absence and presence of Dglucose (6, 7). By means of this method, polypeptides of M, 45,000 to 60,000 have been detected in the human erythrocyte (6, 7), chick embryo fibroblast (8, 9), rat adipocyte (9, lo), and human placenta (11). The second approach is an immunological one. The glucose transporter from human erythrocytes has been purified, and rabbit antisera have been raised against the purified protein (12, 13). These anti-transporter antibodies have been used to detect a cross-reacting polypeptide of M, 55,000 in HeLa cells (13), ilf,. 41,000 in chick embryo fibroblasts (14), and M, 45,000 in rat adipocytes (15, 16). Prior to this study, we have reported that isolated plasma membranes from rat skeletal muscle retain cytochalasin B-inhib198
IDENTIFICATION
OF THE
MUSCLE
itable D-glucose-transport activity (17). Moreover, the membranes possess a set of high-affinity sites to which cytochalasin B binds reversibly and from which it can be displaced by D-glucose (17); these are presumably located on the transporter. In this study, we report the identification of the glucose-transporter polypeptide in this membrane preparation by both of the methods described above. EXPERIMENTAL
PROCEDURES
Plasma nzembmnc prepurc~ttion Plasma membranes were prepared from rat leg and back muscles exactly as we have previously described (17). The membrane fraction that bands at the interface between 0.7 and 0.9 M sucrose, which has the highest specific activity for plasma membrane-marker enzymes and for [*H]cytochalasin B binding (li’), was used throughout this study. When the membranes were trypsinised, the procedure was similar to that described in Ref. (17): membranes (1.2 mg protein in 200 ~1 5 mM Na phosphate buffer, pH 8.0) were treated with 10 pg trypsin for 15 min at room temperature. The reaction was stopped by the addition of 8 ml of buffer containing 0.1 mM phenylmethanesulfonyl fluoride. The digested membranes were washed twice by centrifugation. Protein was estimated by the procedure of Lowry et al. (18). Transpwter-specdlic c@odwhAn B-bindingsites The number of transporter-specific cytochalasin B-binding sites in the plasma membranes was estimated according to the procedure described previously (17). In this procedure, the amounts of PH]cytochalasin B bound to the membranes in the presence of 370 mM L- and n-glucose are measured by means of an ultrafiltration method to separate the medium from the membranes. Cytochalasin E (4 PM) is included throughout in order to prevent the binding of cytochalasin B to high-affinity sites on proteins other than the transporter. The difference between the amounts bound in the presence of L-glucose and of n-glucose is considered to be due to binding to transporterspecific sites. Because only an estimate of the number of sites was required, the measurements were made at a single concentration of THJcytochalasin B. The concentration chosen was 0.5 PM. This concentration was high enough so that about 65% of the high-affinity sites in these membranes (dissociation constant, 0.28 PM (17)) were occupied, but low enough so that there was not overwhelming binding to the low-affinity sites. As a result, the number of transporter sites may be underestimated by as much as 35%. Ph.otokbdingtith[*H]eytochalasinB. Photolabeling of the glucose transporter with [aH]cytochalasin B
GLUCOSE
TRANSPORTER
199
was performed by a modification of recently described procedures (6,7). Muscle membranes were suspended at a protein concentration of 4 to 6 mg/ml in 5 mM sodium phosphate, 250 mM sucrose, 0.6 to 2% ethanol, 1 mM EDTA, pH 7.4, containing 5 p~ cytochalasin E, L- or D-ghCOSe, and mlcytochalasin B (irradiation buffer). The concentrations of the latter three compounds are given in the legend to Fig. 1. The mixtures were irradiated with the 150-W xenon-arc lamp in an Aminco-Bowman spectrofluorometer. Irradiation was either at 280 nm, in which case the sample was at a distance of 12 cm from the lamp and at room temperature, or without wavelength selection, in which case the sample was 8 cm from the lamp at room temperature for 15- to 30-s intervals separated by 30-s periods in ice water. Other details are given in Fig. 1. After irradiation, the membranes were washed once with the phosphate-sucrose buffer containing 10 pM nonradioactive cytochalasin B and twice more in the same buffer without cytochalasin B by centrifugation and resuspension. The labeled membranes were analyzed on slab gels by SDS3-polyacrylamide gel electrophoresis, as described below. After staining of the polypeptides with Coomassie blue, the gels were sliced into l-mm segments. Samples that had been irradiated in the presence of L- and n-glucose were run in adjacent lanes on the gel, and these were always sliced simultaneously. The gel slices were solubilized with 0.6 ml 30% HzOz at 75°C for 16 h and counted in 10 ml Aquasol (New England Nuclear), at an efficiency of 22%. Inamurwlogical procedures. The rabbit antiserum against the purified human erythrocyte glucose transporter used in this study has been previously described (16). Purified human erythrocyte transporter (19) was coupled to Bio-Rad A&Gel 102 and antibodies against the transporter were isolated from the rabbit antiserum on this material according to the procedure of Sogin and Hinkle (13). Rat muscle plasma membranes were subjected to SDS-gel electrophoresis as described below. The procedures for blotting the polypeptides onto nitrocellulose and for treating the nitrocellulose with antibody and with lzI-labeled second antibody were exactly as described previously (16). SDS-gel electruphcn-esis.Two procedures were used for SDS-gel electrophoresis. In one of these, the samples were treated at room temperature with 2% “lauryl sulfate” from Pierce Chemical Company (a mixture of 12-, 14-, and Is-carbon alkyl sulfates)/10 mM dithiothreitol. These were then run on 10% polyacrylamide gels with 5% polyacrylamide stacking gels, with
a Abbreviations used: SDS, sodium dodecyl sulfate; IgG, immunoglobulin G.
200
KLIP
0.1% Pierce lauryl sulfate in the gels and running buffer, according to the method of Laemmli (20). In the other procedure, the samples were treated at room temperature with 5% pure dodecyl sulfate (only the 12-carbon sulfate)/8% 2-mercaptoethanol. They were then subjected to electrophoresis on 5 to 15% polyacrylamide gradient gels, according to the method of Laemmli (20). For the photolabeling experiments, both procedures were used, with slab gels that were 8 cm long and 1.5 mm thick, containing 2.5-cm slots into which up to 500 fig of protein was loaded. For the immunological identification, only the first-described procedure was employed, with slab gels that were 6 cm long and 0.5 mm thick. In this case, the samples were sometimes treated with 30 mM N-ethylmaleimide for 5 min after the lauryl sulfate/dithiothreitol exposure; this treatment did not alter the mobility of the transporter polypeptide. Materids. FHlCytochalasin B (17.4 CVmmol) was obtained from Amersham. RESULTS
Photolabeling of the Muscle Transporter with [SH]Cytochalasin B (a) Molecular weight and o-glucose pm tectim Parallel samples of muscle plasma membranes were exposed to rH]cytochalasin B in the presence of D- or L-glucose, as described under Experimental Procedures and in Fig. 1. After irradiation of the samples and removal of the free ligand, the polypeptides were resolved by SDS-gel electrophresis. Only one region of the gels (enclosed by the arrows in Figs. la-c) showed a peak of radioactivity that was significantly less as the result of irradiation in the presence of D-glucose. On 10% polyacrylamide gels this peak was located just above the position of band 5 of the human erythrocyte membrane (Fig. la), and thus the apparent molecular weight of the labeled polypeptide is 45,000 to 50,000 (see Fig. le for calibration of the 10% gels). For reasons that are given under Discussion, we consider this polypeptide to be the glucose transporter and will refer to it as such hereafter. When labeled membranes were analyzed in 5 to 15% polyacrylamide gradient gels, the mobility of the transporter polypeptide coincided with that of band 6 of the erythrocyte membrane (Fig. lb), and so corresponds to an apparent molecular weight of
ET AL.
38,000 (see Fig. If for calibration of the gradient gels). The percentage by which Dglucose decreased the labeling of this polypeptide, relative to that found with L-glucose, fell in the range of 60 to 85% (Figs. la, b). The irradiation wavelength used in Fig. la was 280 nm. This may account for the lower photolabeling achieved relative to that of Fig. lb in which the unfiltered light from the xenon arc was used.4 In addition to the peak of labeled transporter, a peak of labeling that was not inhibited by D-g]UCOSe occurred in the region of the gel corresponding to M, 100,000 (Figs. la, b). This peak coincided with the location of a major polypeptide component in the plasma membranes (Figs. la, b). We have found that mild trypsin treatment of the membranes led to the disappearance of the Coomassie blue-stained band at 100,000, but did not decrease the level of reversible, D-glucose-inhibitable [3H]cytochalasin B binding to the membranes (Ref. (17) and Table I). The effect of prior trypsinization on the photolabeling was examined by irradiating the trypsin-treated membranes with [3H]cytochalasin B. The results in Fig. lc show that the proteolytic treatment selectively abolished the radioactive peak in the M, 100,000 region, but did not significantly alter the location of the transporter peak. Thus, in agreement with the expectation from the results of reversible [3H]cytochalasin B binding, mild trypsinization does not appear to cleave the transporter. In order to test the specificity of the labeling of the putative transporter, two types of control experiments were performed. In one, the buffer for irradiation was 5 mM sodium phosphate, 250 mM sucrose pH 5. After treatment at this pH, no reversible D-glucose-inhibitable binding of [‘Hlcytochalasin B to the membranes can be detected at pH 7.4, and no D-glucoseprotectable photolabeling occurred (Fig. 4 Because [8H]CB does not absorb light at 280 nm, the results of Fig. la suggest that aromatic side chains of the membrane protein may be the photoactivated species in the labeling reaction (M. P. Czech, personal communication; see also A. Klip, and D. Walker, (1983) Biophys. J. 41,185a).
IDENTIFICATION
OF THE MUSCLE GLUCOSE TRANSPORTER
0
05
10
Rf
0
6
10
20 0’2
30 0’4
40
50 06
I A‘ O’S
FIG. 1. Photolabeling of muscle membranes with vH]cytochalasin B. (a) Native membranes on 10% gels. Photolabeling of 2.5 mg membrane protein in 456 pl was carried out at 230 nm for a period of 25 min, with five additions of 0.15 PM [~@ytocbalasin B made at 5-min intervals, in the presence of 0.39 M L (@)- or D (0)-glucose. After removal of the free THJcytochalasin B, 500 fig of protein was loaded in each well of the 10% gels and processed as described under Experimental Procedures. The units of the abscissa are slice number and relative migration with reference to the position of bromphenol blue. The vertical arrows denote the limit of n-glucose-inhibitable labeling here and in (b) and (c). (b) Native membranes on gradient gels. Photolabeling of 2.5 mg membrane proteins in 450 ~1was performed with three additions of 0.5 PM [SH]cytochalasin B, each of which was followed by 15 s of irradiation without wavelength selection of the xenon arc. Mixtures contained either 0.22 M L (a)- or D (0)-glucose; 400 pg of protein was loaded in each well of the gradient gels. (c) Trypsin-treated membranes on gradient gels. Muscle membranes were treated with trypsin as described under Experimental Procedures, prior to photolabeling of 1.6 mg in 400 ~1,in the presence of 0.39 M L (a)- or D (0)-glucose. Five additions of [aH)eytochalasin B were made, each was followed by 30 s of irradiation without wavelength selection of the xenon arc; 315 pg of protein was loaded in each well. (d) Membranes at low pH on gradient gels. Muscle membranes were suspended in irradiation buffer at pH 5. Irradiation was performed for a 2-min interval after each of three successive additions of 0.5 @Y [8H]cytochalasin B. Other conditions as in (b). (e) Calibration of 10% gels. The logarithms of M, of the major polypeptides of the human erythrocyte membrane are plotted against their migration relative to that of bromphenol blue on 10% polyacrylamide gels. The numbers beside the points are the nomenclature of the polypeptides according to Steck (24), and the values of M, are also from (24). The bar designates the migration position of the n-glucose-inhibited peak of label shown in (a). (f) Calibration of gradient gels. Human erythrocyte membrane polypeptides were separated on 5 to 15% polyacrylamide gradient gels; other details are as in (e). The bar designates the migration position of the D-glucose-inhibited peak of label shown in (b).
202
KLIP
ET AL.
TABLE
I
REVERSIBLEBINDINGAND PHOTOACPIVATED COVALENTATTACHMENTOF@IJCYTOCHALASINB TO THE GLUCOSE TRANSPORTER IN MUSCLEPLASMAMEMBRANES Total D-ghCOSeinhibitable CBbinding sitesa
CB covalently bound*
1. Unirradiated Irradiated
1.9 1.9”
0.016
0.8
2. Unirradiated Irradiated
2.0 n.d.d
0.06
3.0
3. Not trypsinized, unirradiated Trypsinized, unirradiated Trypsinized, irradiated
2.1 2.7 n.d.d
0.18
6.6
Sample
Percentage photolabeling
a Determined as described under Experimental Procedures. CB refers to cytochalasin B. Results are expressed as pmol/mg protein. *Calculated from the difference between the amounts of label found in the transporter peak after irradiation in the presence of L- and D-ghCOSe. Experiments 1,2, and 3 are those shown in Figs. la, b, and c, respectively. c Cytochalasin B binding was measured after irradiation and washing of the membranes. The small amount of covalently bound label did not interfere with this determination. dNot determined.
Id). The fact that D-glucose-insensitive labeling of the lOO,OOO-Da component did occur indicates that the photoreactivity of [‘HJcytochalasin B was not impaired at pH 5. In the other control experiment, [‘Hlcytochalasin B was irradiated in the absence of membranes and then added back to the membrane suspension. When this sample was subjected to electrophoresis, no radioactivity was found in the region corresponding to the Mr range between 200,000 and 20,000. (b) Extent of photo~ling. To determine the extent of photolabeling of the glucose transporter, we compared the amount of D-glucose-inhibitable, covalently bound PlXjcytochalasin B found in the transporter peak after irradiation in the presence of L-glucose with the maximum amount of [8H]cytochalasin B reversibly bound to transporter sites in the membranes at equilibrium (Table I). Only a few percent of the total D-glucose-inhibitable cytochalasin B-binding sites were covalently labeled. In order to ascertain whether this low percentage of photolabeling resulted from destruction of the cytochalasin Bbinding site due to irradiation, the reversible binding of [3H]cytochalasin B at equilibrium was also measured after the
membranes had been photolabeled in the presence of [3H]cytochalasin B and L-glucose. The results in Table I show that irradiation did not significantly decrease the amount of transporter-specific cytochalasin B-binding sites. In an effort to obtain a higher yield for the covalent incorporation of rH]cytochalasin B, several conditions for photolysis were examined with human erythrocytes membranes, which are more readily available and in which the glucose transporter was originally photolabeled (6, 7). The procedures examined were (i) 6min irradiation with the xenon lamp without wavelength selection, with 0.8 pM [3H]cytochalasin B; (ii) 60-min irradiation at 280 nm with 1.4 PM rH]cytochalasin B; and (iii) lOO-min irradiation at 280 nm with successive additions of 0.5 pM rH]cytochalasin B every 20 min. These conditions were designed to examine the effect on yield of (i) radiation that included light of the wavelength of high absorbance of cytochalasin B (below 210 nm), (ii) prolonged irradiation at the wavelength of maximum protein absorbance, and (iii) multiple additions of the ligand, made on the supposition that extensive photoactivated destruction of it might be occurring. The ir-
IDENTIFICATION
OF THE
MUSCLE
GLUCOSE
TRANSPORTER
203
less than 2% of the total transporter sites (6, 7). Immun&gical Identification of the Muscle Transporter - 100 Rabbit antibodies against the purified glucose transporter from human erythrocytes were isolated from serum by affinity chromatography on the immobilized human erythrocyte transporter. These an- 58 tibodies were then used to detect any re- 53 active polypeptide in the rat muscle plasma membranes. The polypeptides of the mus- 37 cle membrane were separated by SDS-gel electrophoresis and then transferred to nitrocellulose. This blot was treated sequen- 31 tially either with the affinity-purified antibody against the transporter or with nonimmune rabbit IgG and then with ‘%Ilabeled goat antibody against rabbit IgG. Figure 2 presents the autoradiograms of FIG. 2. Autoradiogram of nitrocellulose blots of rat the blots. A polypeptide with a mobility muscle plasma membrane polypeptides. Rat muscle corresponding to a molecular weight of plasma membranes (26 fig protein per lane) were subjected to SDS-gel electrophoresis, transferred to niabout 51,000 is seen to have reacted with trocellulose, and treated with either 160 pg of purified the anti-transporter antibody, whereas antibody against the human erythrocyte transporter there was no reaction with nonimmune (lane A) or 160 pg of nonimmune rabbit IgG (lane rabbit IgG. A repeat of this experiment B). The positions of standard proteins, which were gave identical results, with the exception run in an adjacent lane on the gel, transferred to that the average mobility of the labeled nitrocellulose, and detected by staining, are indicated band was at M, 48,500; we attribute the by their molecular weight in thousands. The standards small difference to imprecision in the prowere, in order of decreasing molecular weight, rabbit cedure. muscle phosphorylase, beef liver catalase, glutamic During the course of this study, it was dehydrogenase, yeast alcohol dehydrogenase, and pig heart lactic dehydrogenase (25, 26). found that the method of sample preparation for SDS-gel electrophoresis affected the electrophoretic behavior of the transporter. When the sample was held at 100°C radiations were performed in the presence for 2 min, rather than simply at room temof 0.39 M D- or L-glucose, and the mem- perature, the transporter polypeptide mibranes were analyzed by SDS-gel electro- grated at M, 45,000 (Fig. 3). Moreover, there phoresis as described above. The peak of was a decrease in the intensity of this band labeled erythrocyte transporter occurred and the corresponding appearance of sevin the M, 58,000 to 48,000 region of the gels. eral labeled bands of higher molecular None of the conditions led to extensive co- weight (Fig. 3). Presumably, the latter efvalent labeling. The amounts of D-glucose- fect is due to partial aggregation of the protected incorporation of [3H]cytochalasin transporter, either with itself or other B were equivalent to 1.0,2.3, and 8.4 pmol/ polypeptides. mg protein applied to the gel for conditions i, ii, and iii, respectively. These values are Comparison with the Rat Adipocyte Transporter in the range of values (3 to 35 pmol/mg) found under slightly different conditions Previously, the same antiserum emduring the original photolabeling of the erythrocyte transporter and correspond to ployed in this study was used to detect the
204
KLIP ET AL.
photolabeling and immunological procedures to be the glucose transporter for the following reasons:
- 58 - 53 - 37 - 31
FIG. 3. The effect of sample preparation upon the autoradiogram pattern of the rat muscle plasma membranes. Samples were prepared for SDS-gel electrophoresis according to the procedure given under Experimental Procedures (no heating, lane A) or with the variation that the sample was held at 100°C for 2 min (lane B). Other conditions were as described in Fig. 2. The single nitrocellulose blot was treated with 160 fig of affinity-purified antibody.
glucose transporter in plasma membranes and microsomes from basal and insulinstimulated rat adipocytes (16). In that study, the samples for SDS-gel electrophoresis were heated at 100°C for 2 min, and the adipocyte transporter was identified in all the fractions as a 45,000-Da polypeptide. In order to compare the transporters from the two rat tissues directly, we have run heated and unheated SDS samples of plasma membranes from rat muscle and fat cells in a single gel. The mobilities of the transporters from the two tissues were identical after both methods of sample preparation (data not shown); thus the apparent molecular weight of the adipocyte transporter is also greater when the sample is not heated. DISCUSSION
We consider the polypeptide of molecular weight 45,000 to 50,000 identified by the
(i) The polypeptide is more extensively photolabeled by [3H]cytochalasin B in the presence of L- than D-ghCOSe. D-Glucose competitively inhibits the binding of [3H]cytochalasin B to the purified glucose transporter from human erythrocytes, whereas L-glucose does not (21). Although actin, which has a molecular weight of about 45,000, is also known to be photolabeled with [3H]cytochalasin B, D-glucose does not inhibit this labeling (6, 7, 22). (ii) The putative transporter polypeptide reacts with rabbit antibody against the purified transporter from human erythrocytes but not with control rabbit IgG. It could be argued that this reaction is fortuitous. However, the identification is strengthened by the fact that the muscle polypeptide has the same electrophoretic mobility as that of the adipocyte transporter previously identified with the same antiserum. In the case of the adipocyte, the relative amounts of the transporter polypeptide detected in various membrane fractions (plasma membranes and microsomes from basal and insulin-treated cells) by the immunological method were found to be in approximate agreement with the relative amounts of transporter measured by the binding of cytochalasin B (15,16). (iii) The apparent molecular weight of the muscle polypeptide is virtually the same as that of the purified glucose transporter from human erythrocytes. After partial removal of its extensive carbohydrate with glycosidase, the erythrocyte polypeptide runs as a species of molecular weight 46,000 on SDS-gel electrophoresis when the sample is heated and the gel is 10% polyacrylamide (19). In the case of the erythrocyte transporter, it has been shown that the purified protein exhibits the transport function in a reconstituted membrane (22, 23), and thus appears to constitute the entire transport system. The migration of the muscle transporter upon SDS-gel electrophoresis, relative to the molecular weight standards, which were largely water-soluble proteins, was
IDENTIFICATION
OF THE
MUSCLE
found to be sensitive to both the electrophoretic conditions (Fig. 1) and the method of sample preparation (Fig. 3). The reasons for these changes in relative mobility are unknown, but since the transporter is an integral membrane protein, their occurrence is not unexpected. The apparent molecular weight of the partially deglycosylated erythrocyte transporter varies from 43,000 on 7.5% acrylamide gels to 50,000 on 12.5% gels (19). For the purposes of purifying the muscle transporter and investigating its regulation it would be advantageous to achieve complete photolabeling with rH]cytochalasin B. However, for reasons that are not known, in none of the cell types in which the transporter has been photolabeled has more than a few percent labeling been obtained. In this regard, it is worth noting that the amounts of incorporated label may therefore not provide an accurate measure of the relative amounts of transporter under various conditions. Nevertheless, the availability of two methods to identify the muscle transporter should facilitate investigation of the molecular bases of the various types of regulation. Note added in proo$ Recently, the number of transporter-specific cytochalasin B binding sites was determined by the centrifugation procedure of reference (16). The values found are about B-fold larger than those reported herein, which were determined by an ultrafiltration assay. Evidently, a large fraction of the cytochalasin B bound to the transporter is lost during the wash of the filter. ACKNOWLEDGMENTS This research was supported by grants from the Medical Research Council of Canada to A.K. and from the National Institutes of Health to G.E.L. (GM 22996 and AM 25336). A.K. is an M.R.C. Scholar. D.S. is supported by a fellowship from the Juvenile Diabetes Foundation (No. 82F084). We are grateful to Dr. S. Grinstein and Dr. M. P. Czech for helpful suggestions concerning the photolabeling procedure, and to S. A. Olson and Dr. M. J. Weber for preparing the antiserum.
REFERENCES 1. CLAUSEN, T. (1975) Curr. Top. Membrane Tramp. 6, 169-226. 2. ELBRINCK, I., AND BIHLER, I. (1975) Soienoe 188, 1177-1184.
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TRANSPORTER
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3. KLIP, A., LOGAN, W. J., ANDLI, G. (1982) B&him Biophys. Actu 682,265-280. 4. WARDZALA, L. J., AND JEANRENAUD, B. (1981) J. Bid Chzem 256, 7090-7093. 5. GARTWAITE, S. M., AND HOLLOSZY, J. 0. (1982) J. Biol Chem 257, 5008-5012. 6. CARTER-SU, C., PESSIN, J. E., MORA, R., GITOMER, W., AND CZECH, M. P. (1982) J. Biol Chem. 257, 54165425. 7. SHANAHAN, M. F. (1982) J. Biol Chxm. 257,72907293. 8. PESSIN, J. E., TILLOTSON, L. G., YAMADA, K., GITOMER, W., CARTER-SU, C., MORA, R., ISSELBACHER, K. J., AND CZECH, M. P. (1982) Proc Natl Acad Sci USA 79,2286-2290. 9. SHANAHAN, M. F., OLSON, S. A., WEBER, M. J., LIENHARD, G. E., AND GORGA, J. C. (1982) Biochem. Biophys. Res. Ccnnmun 107,38-43. 10. PESSIN, J. E., GITOMER, W., AND CZECH, M. P. (1982) Dinbeti 31,29A. 11. JOHNSON, L. W., AND SMITH, C. H. (1982) Biochem. Biophys. Res. Commun. 109, 408-413. 12. BALDWIN, S. A., AND LIENHARD, G. E. (1980) Biochem Bisphys. Res Commun 94.1401-1408. 13. SOGIN, D. C., AND HINKLE, P. C. (1980) Proc. Natl. Acad Sci USA 77, 5725-5729. 14. SALTER, D. W., BALDWIN, S. A., LIENHARD, G. E., AND WEBER, M. J. (1982) Proc. Natl. Acad Sci USA 79, X40-1544. 15. WHEELER, T. J., SIMPSON, I. A., SOGIN, D. C., HINKLE, P. C., AND CUSHMAN, S. W. (1982) Biochem. Biophys. Res. Commun 105,89-95. 16. LIENHARD, G. E., KIM, H. H., RANSOME, K. J., AND GORGA, J. C. (1982) Bioch.em. Biophys. Res. Commun 105, 1150-1156. 17. KLIP, A., AND WALKER, D. (1983) Arch Biochem Biophys. 221, 175-187. 18. LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. L., AND RANDALL, R. J. (1951) J. Biol &em. 193, 265-275. 19. BALDWIN, S. A., BALDWIN, J. M., AND LIENHARD, G. E. (1982) Biochemistry 21.3836-3842. 20. LAEMMLI, U. K. (1970) Nature (Lo&on) 227,681685. 21. BALDWIN, S. A., BALDWIN, J. M., GORGA, F. R., AND LIENHARD, J. E. (1979) B&him Babphys. Ada 552, X33-188. 22. BALDWIN, J. M., GORGA, J. C., AND LIENHARD, G. E. (1981) J. Biol Chem 256,3685-3689. 23. WHEELER, T. J., AND HINKLE, P. C. (1981) J. Biol Owm. 256,8907-8914. 24. STECK, T. L. (1974) J. Cell Biol 62, 1-19. 25. WEBER, K., AND OSBORNE, M. (1975) in The Proteins (Neurath, H., and Hill, R. L., eds.), Vol. 1, pp. 179-223, Academic Press, New York. 26. AMES, GF.-L., ANDNIKAIDO, K. (1975) Bio&emist~ 15. 616-623.
Identification
of the Glucose Transporter
in Rat Skeletal Muscle’
AMIRA KLIP,2 DENISE WALKER, KATHLEEN J. RANSOME,* DEAN W. SCHROER,* AND GUSTAV E. LIENHARD* Division of Neurology, Research Institute, Canada, and *Department of Biochemistry,
The Hospital for Sick Children, Tunmto, Ontario M5G lx& Dartmouth Medical school, Hanover, New Hampshire OS756
Received March 7, 1983, and in revised form May 27, 1983
The glucose transporter in the plasma membrane of rat skeletal muscle has been identified by two approaches. In one, the transporter was detected as the polypeptide that was differentially labeled by photolysis with [‘Hlcytochalasin B in the presence of L- and D-glucose. [‘HlCytochalasin B is a high-affinity ligand for the transporter that is displaced by D-glucose. In the other, the transporter was detected by means of its reaction with rabbit antibodies against the purified glucose transporter from human erythrocytes. By both procedures, the transporter was found to be a polypeptide with a mobility corresponding to a molecular weight of 45,000-50,000 upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis.
Glucose transport into skeletal muscle cells occurs through a system of the facilitated diffusion type (l-3). This system exhibits several kinds of regulation. Transport is stimulated by the hormones insulin and epinephrine, by contractile activity, and by anoxia (1,2). A recent study has shown that insulin probably enhances glucose transport in muscle by causing the translocation of transporters from an intracellular location to the plasma membrane (4). The bases for the other types of regulation are not known. The actions of epinephrine and contractile activity of muscle differ in part from those of insulin, since the reversal of the stimulation by epinephrine and contractile activity requires protein synthesis, whereas the reversal of the insulin effect does not (5). The glucose-transporter protein in muscle has not been previously identified. Further investigation of these regulatory phenomena would be facilitated by such identification. Recently, two approaches have 1 This paper is the second in a series. No. 1 is Ref. (17). 2 To whom correspondence should be addressed. 0003-9861/83 $3.00 Copyright All rights
0 1983 by Academic Press, Inc. of reproduction in any form reserved.
proven successful in the detection of the glucose transporter in other cell types. In one of these, the transporter was photoaffinity-labeled with [‘Hlcytochalasin B, a high-affinity inhibitor of the transporter. D-Glucose competes with cytochalasin B for reversible binding to the transporter; hence the transporter polypeptide can be detected by its differential incorporation of label in the absence and presence of Dglucose (6, 7). By means of this method, polypeptides of M, 45,000 to 60,000 have been detected in the human erythrocyte (6, 7), chick embryo fibroblast (8, 9), rat adipocyte (9, lo), and human placenta (11). The second approach is an immunological one. The glucose transporter from human erythrocytes has been purified, and rabbit antisera have been raised against the purified protein (12, 13). These anti-transporter antibodies have been used to detect a cross-reacting polypeptide of M, 55,000 in HeLa cells (13), ilf,. 41,000 in chick embryo fibroblasts (14), and M, 45,000 in rat adipocytes (15, 16). Prior to this study, we have reported that isolated plasma membranes from rat skeletal muscle retain cytochalasin B-inhib198
IDENTIFICATION
OF THE
MUSCLE
itable D-glucose-transport activity (17). Moreover, the membranes possess a set of high-affinity sites to which cytochalasin B binds reversibly and from which it can be displaced by D-glucose (17); these are presumably located on the transporter. In this study, we report the identification of the glucose-transporter polypeptide in this membrane preparation by both of the methods described above. EXPERIMENTAL
PROCEDURES
Plasma nzembmnc prepurc~ttion Plasma membranes were prepared from rat leg and back muscles exactly as we have previously described (17). The membrane fraction that bands at the interface between 0.7 and 0.9 M sucrose, which has the highest specific activity for plasma membrane-marker enzymes and for [*H]cytochalasin B binding (li’), was used throughout this study. When the membranes were trypsinised, the procedure was similar to that described in Ref. (17): membranes (1.2 mg protein in 200 ~1 5 mM Na phosphate buffer, pH 8.0) were treated with 10 pg trypsin for 15 min at room temperature. The reaction was stopped by the addition of 8 ml of buffer containing 0.1 mM phenylmethanesulfonyl fluoride. The digested membranes were washed twice by centrifugation. Protein was estimated by the procedure of Lowry et al. (18). Transpwter-specdlic c@odwhAn B-bindingsites The number of transporter-specific cytochalasin B-binding sites in the plasma membranes was estimated according to the procedure described previously (17). In this procedure, the amounts of PH]cytochalasin B bound to the membranes in the presence of 370 mM L- and n-glucose are measured by means of an ultrafiltration method to separate the medium from the membranes. Cytochalasin E (4 PM) is included throughout in order to prevent the binding of cytochalasin B to high-affinity sites on proteins other than the transporter. The difference between the amounts bound in the presence of L-glucose and of n-glucose is considered to be due to binding to transporterspecific sites. Because only an estimate of the number of sites was required, the measurements were made at a single concentration of THJcytochalasin B. The concentration chosen was 0.5 PM. This concentration was high enough so that about 65% of the high-affinity sites in these membranes (dissociation constant, 0.28 PM (17)) were occupied, but low enough so that there was not overwhelming binding to the low-affinity sites. As a result, the number of transporter sites may be underestimated by as much as 35%. Ph.otokbdingtith[*H]eytochalasinB. Photolabeling of the glucose transporter with [aH]cytochalasin B
GLUCOSE
TRANSPORTER
199
was performed by a modification of recently described procedures (6,7). Muscle membranes were suspended at a protein concentration of 4 to 6 mg/ml in 5 mM sodium phosphate, 250 mM sucrose, 0.6 to 2% ethanol, 1 mM EDTA, pH 7.4, containing 5 p~ cytochalasin E, L- or D-ghCOSe, and mlcytochalasin B (irradiation buffer). The concentrations of the latter three compounds are given in the legend to Fig. 1. The mixtures were irradiated with the 150-W xenon-arc lamp in an Aminco-Bowman spectrofluorometer. Irradiation was either at 280 nm, in which case the sample was at a distance of 12 cm from the lamp and at room temperature, or without wavelength selection, in which case the sample was 8 cm from the lamp at room temperature for 15- to 30-s intervals separated by 30-s periods in ice water. Other details are given in Fig. 1. After irradiation, the membranes were washed once with the phosphate-sucrose buffer containing 10 pM nonradioactive cytochalasin B and twice more in the same buffer without cytochalasin B by centrifugation and resuspension. The labeled membranes were analyzed on slab gels by SDS3-polyacrylamide gel electrophoresis, as described below. After staining of the polypeptides with Coomassie blue, the gels were sliced into l-mm segments. Samples that had been irradiated in the presence of L- and n-glucose were run in adjacent lanes on the gel, and these were always sliced simultaneously. The gel slices were solubilized with 0.6 ml 30% HzOz at 75°C for 16 h and counted in 10 ml Aquasol (New England Nuclear), at an efficiency of 22%. Inamurwlogical procedures. The rabbit antiserum against the purified human erythrocyte glucose transporter used in this study has been previously described (16). Purified human erythrocyte transporter (19) was coupled to Bio-Rad A&Gel 102 and antibodies against the transporter were isolated from the rabbit antiserum on this material according to the procedure of Sogin and Hinkle (13). Rat muscle plasma membranes were subjected to SDS-gel electrophoresis as described below. The procedures for blotting the polypeptides onto nitrocellulose and for treating the nitrocellulose with antibody and with lzI-labeled second antibody were exactly as described previously (16). SDS-gel electruphcn-esis.Two procedures were used for SDS-gel electrophoresis. In one of these, the samples were treated at room temperature with 2% “lauryl sulfate” from Pierce Chemical Company (a mixture of 12-, 14-, and Is-carbon alkyl sulfates)/10 mM dithiothreitol. These were then run on 10% polyacrylamide gels with 5% polyacrylamide stacking gels, with
a Abbreviations used: SDS, sodium dodecyl sulfate; IgG, immunoglobulin G.
200
KLIP
0.1% Pierce lauryl sulfate in the gels and running buffer, according to the method of Laemmli (20). In the other procedure, the samples were treated at room temperature with 5% pure dodecyl sulfate (only the 12-carbon sulfate)/8% 2-mercaptoethanol. They were then subjected to electrophoresis on 5 to 15% polyacrylamide gradient gels, according to the method of Laemmli (20). For the photolabeling experiments, both procedures were used, with slab gels that were 8 cm long and 1.5 mm thick, containing 2.5-cm slots into which up to 500 fig of protein was loaded. For the immunological identification, only the first-described procedure was employed, with slab gels that were 6 cm long and 0.5 mm thick. In this case, the samples were sometimes treated with 30 mM N-ethylmaleimide for 5 min after the lauryl sulfate/dithiothreitol exposure; this treatment did not alter the mobility of the transporter polypeptide. Materids. FHlCytochalasin B (17.4 CVmmol) was obtained from Amersham. RESULTS
Photolabeling of the Muscle Transporter with [SH]Cytochalasin B (a) Molecular weight and o-glucose pm tectim Parallel samples of muscle plasma membranes were exposed to rH]cytochalasin B in the presence of D- or L-glucose, as described under Experimental Procedures and in Fig. 1. After irradiation of the samples and removal of the free ligand, the polypeptides were resolved by SDS-gel electrophresis. Only one region of the gels (enclosed by the arrows in Figs. la-c) showed a peak of radioactivity that was significantly less as the result of irradiation in the presence of D-glucose. On 10% polyacrylamide gels this peak was located just above the position of band 5 of the human erythrocyte membrane (Fig. la), and thus the apparent molecular weight of the labeled polypeptide is 45,000 to 50,000 (see Fig. le for calibration of the 10% gels). For reasons that are given under Discussion, we consider this polypeptide to be the glucose transporter and will refer to it as such hereafter. When labeled membranes were analyzed in 5 to 15% polyacrylamide gradient gels, the mobility of the transporter polypeptide coincided with that of band 6 of the erythrocyte membrane (Fig. lb), and so corresponds to an apparent molecular weight of
ET AL.
38,000 (see Fig. If for calibration of the gradient gels). The percentage by which Dglucose decreased the labeling of this polypeptide, relative to that found with L-glucose, fell in the range of 60 to 85% (Figs. la, b). The irradiation wavelength used in Fig. la was 280 nm. This may account for the lower photolabeling achieved relative to that of Fig. lb in which the unfiltered light from the xenon arc was used.4 In addition to the peak of labeled transporter, a peak of labeling that was not inhibited by D-g]UCOSe occurred in the region of the gel corresponding to M, 100,000 (Figs. la, b). This peak coincided with the location of a major polypeptide component in the plasma membranes (Figs. la, b). We have found that mild trypsin treatment of the membranes led to the disappearance of the Coomassie blue-stained band at 100,000, but did not decrease the level of reversible, D-glucose-inhibitable [3H]cytochalasin B binding to the membranes (Ref. (17) and Table I). The effect of prior trypsinization on the photolabeling was examined by irradiating the trypsin-treated membranes with [3H]cytochalasin B. The results in Fig. lc show that the proteolytic treatment selectively abolished the radioactive peak in the M, 100,000 region, but did not significantly alter the location of the transporter peak. Thus, in agreement with the expectation from the results of reversible [3H]cytochalasin B binding, mild trypsinization does not appear to cleave the transporter. In order to test the specificity of the labeling of the putative transporter, two types of control experiments were performed. In one, the buffer for irradiation was 5 mM sodium phosphate, 250 mM sucrose pH 5. After treatment at this pH, no reversible D-glucose-inhibitable binding of [‘Hlcytochalasin B to the membranes can be detected at pH 7.4, and no D-glucoseprotectable photolabeling occurred (Fig. 4 Because [8H]CB does not absorb light at 280 nm, the results of Fig. la suggest that aromatic side chains of the membrane protein may be the photoactivated species in the labeling reaction (M. P. Czech, personal communication; see also A. Klip, and D. Walker, (1983) Biophys. J. 41,185a).
IDENTIFICATION
OF THE MUSCLE GLUCOSE TRANSPORTER
0
05
10
Rf
0
6
10
20 0’2
30 0’4
40
50 06
I A‘ O’S
FIG. 1. Photolabeling of muscle membranes with vH]cytochalasin B. (a) Native membranes on 10% gels. Photolabeling of 2.5 mg membrane protein in 456 pl was carried out at 230 nm for a period of 25 min, with five additions of 0.15 PM [~@ytocbalasin B made at 5-min intervals, in the presence of 0.39 M L (@)- or D (0)-glucose. After removal of the free THJcytochalasin B, 500 fig of protein was loaded in each well of the 10% gels and processed as described under Experimental Procedures. The units of the abscissa are slice number and relative migration with reference to the position of bromphenol blue. The vertical arrows denote the limit of n-glucose-inhibitable labeling here and in (b) and (c). (b) Native membranes on gradient gels. Photolabeling of 2.5 mg membrane proteins in 450 ~1was performed with three additions of 0.5 PM [SH]cytochalasin B, each of which was followed by 15 s of irradiation without wavelength selection of the xenon arc. Mixtures contained either 0.22 M L (a)- or D (0)-glucose; 400 pg of protein was loaded in each well of the gradient gels. (c) Trypsin-treated membranes on gradient gels. Muscle membranes were treated with trypsin as described under Experimental Procedures, prior to photolabeling of 1.6 mg in 400 ~1,in the presence of 0.39 M L (a)- or D (0)-glucose. Five additions of [aH)eytochalasin B were made, each was followed by 30 s of irradiation without wavelength selection of the xenon arc; 315 pg of protein was loaded in each well. (d) Membranes at low pH on gradient gels. Muscle membranes were suspended in irradiation buffer at pH 5. Irradiation was performed for a 2-min interval after each of three successive additions of 0.5 @Y [8H]cytochalasin B. Other conditions as in (b). (e) Calibration of 10% gels. The logarithms of M, of the major polypeptides of the human erythrocyte membrane are plotted against their migration relative to that of bromphenol blue on 10% polyacrylamide gels. The numbers beside the points are the nomenclature of the polypeptides according to Steck (24), and the values of M, are also from (24). The bar designates the migration position of the n-glucose-inhibited peak of label shown in (a). (f) Calibration of gradient gels. Human erythrocyte membrane polypeptides were separated on 5 to 15% polyacrylamide gradient gels; other details are as in (e). The bar designates the migration position of the D-glucose-inhibited peak of label shown in (b).
202
KLIP
ET AL.
TABLE
I
REVERSIBLEBINDINGAND PHOTOACPIVATED COVALENTATTACHMENTOF@IJCYTOCHALASINB TO THE GLUCOSE TRANSPORTER IN MUSCLEPLASMAMEMBRANES Total D-ghCOSeinhibitable CBbinding sitesa
CB covalently bound*
1. Unirradiated Irradiated
1.9 1.9”
0.016
0.8
2. Unirradiated Irradiated
2.0 n.d.d
0.06
3.0
3. Not trypsinized, unirradiated Trypsinized, unirradiated Trypsinized, irradiated
2.1 2.7 n.d.d
0.18
6.6
Sample
Percentage photolabeling
a Determined as described under Experimental Procedures. CB refers to cytochalasin B. Results are expressed as pmol/mg protein. *Calculated from the difference between the amounts of label found in the transporter peak after irradiation in the presence of L- and D-ghCOSe. Experiments 1,2, and 3 are those shown in Figs. la, b, and c, respectively. c Cytochalasin B binding was measured after irradiation and washing of the membranes. The small amount of covalently bound label did not interfere with this determination. dNot determined.
Id). The fact that D-glucose-insensitive labeling of the lOO,OOO-Da component did occur indicates that the photoreactivity of [‘HJcytochalasin B was not impaired at pH 5. In the other control experiment, [‘Hlcytochalasin B was irradiated in the absence of membranes and then added back to the membrane suspension. When this sample was subjected to electrophoresis, no radioactivity was found in the region corresponding to the Mr range between 200,000 and 20,000. (b) Extent of photo~ling. To determine the extent of photolabeling of the glucose transporter, we compared the amount of D-glucose-inhibitable, covalently bound PlXjcytochalasin B found in the transporter peak after irradiation in the presence of L-glucose with the maximum amount of [8H]cytochalasin B reversibly bound to transporter sites in the membranes at equilibrium (Table I). Only a few percent of the total D-glucose-inhibitable cytochalasin B-binding sites were covalently labeled. In order to ascertain whether this low percentage of photolabeling resulted from destruction of the cytochalasin Bbinding site due to irradiation, the reversible binding of [3H]cytochalasin B at equilibrium was also measured after the
membranes had been photolabeled in the presence of [3H]cytochalasin B and L-glucose. The results in Table I show that irradiation did not significantly decrease the amount of transporter-specific cytochalasin B-binding sites. In an effort to obtain a higher yield for the covalent incorporation of rH]cytochalasin B, several conditions for photolysis were examined with human erythrocytes membranes, which are more readily available and in which the glucose transporter was originally photolabeled (6, 7). The procedures examined were (i) 6min irradiation with the xenon lamp without wavelength selection, with 0.8 pM [3H]cytochalasin B; (ii) 60-min irradiation at 280 nm with 1.4 PM rH]cytochalasin B; and (iii) lOO-min irradiation at 280 nm with successive additions of 0.5 pM rH]cytochalasin B every 20 min. These conditions were designed to examine the effect on yield of (i) radiation that included light of the wavelength of high absorbance of cytochalasin B (below 210 nm), (ii) prolonged irradiation at the wavelength of maximum protein absorbance, and (iii) multiple additions of the ligand, made on the supposition that extensive photoactivated destruction of it might be occurring. The ir-
IDENTIFICATION
OF THE
MUSCLE
GLUCOSE
TRANSPORTER
203
less than 2% of the total transporter sites (6, 7). Immun&gical Identification of the Muscle Transporter - 100 Rabbit antibodies against the purified glucose transporter from human erythrocytes were isolated from serum by affinity chromatography on the immobilized human erythrocyte transporter. These an- 58 tibodies were then used to detect any re- 53 active polypeptide in the rat muscle plasma membranes. The polypeptides of the mus- 37 cle membrane were separated by SDS-gel electrophoresis and then transferred to nitrocellulose. This blot was treated sequen- 31 tially either with the affinity-purified antibody against the transporter or with nonimmune rabbit IgG and then with ‘%Ilabeled goat antibody against rabbit IgG. Figure 2 presents the autoradiograms of FIG. 2. Autoradiogram of nitrocellulose blots of rat the blots. A polypeptide with a mobility muscle plasma membrane polypeptides. Rat muscle corresponding to a molecular weight of plasma membranes (26 fig protein per lane) were subjected to SDS-gel electrophoresis, transferred to niabout 51,000 is seen to have reacted with trocellulose, and treated with either 160 pg of purified the anti-transporter antibody, whereas antibody against the human erythrocyte transporter there was no reaction with nonimmune (lane A) or 160 pg of nonimmune rabbit IgG (lane rabbit IgG. A repeat of this experiment B). The positions of standard proteins, which were gave identical results, with the exception run in an adjacent lane on the gel, transferred to that the average mobility of the labeled nitrocellulose, and detected by staining, are indicated band was at M, 48,500; we attribute the by their molecular weight in thousands. The standards small difference to imprecision in the prowere, in order of decreasing molecular weight, rabbit cedure. muscle phosphorylase, beef liver catalase, glutamic During the course of this study, it was dehydrogenase, yeast alcohol dehydrogenase, and pig heart lactic dehydrogenase (25, 26). found that the method of sample preparation for SDS-gel electrophoresis affected the electrophoretic behavior of the transporter. When the sample was held at 100°C radiations were performed in the presence for 2 min, rather than simply at room temof 0.39 M D- or L-glucose, and the mem- perature, the transporter polypeptide mibranes were analyzed by SDS-gel electro- grated at M, 45,000 (Fig. 3). Moreover, there phoresis as described above. The peak of was a decrease in the intensity of this band labeled erythrocyte transporter occurred and the corresponding appearance of sevin the M, 58,000 to 48,000 region of the gels. eral labeled bands of higher molecular None of the conditions led to extensive co- weight (Fig. 3). Presumably, the latter efvalent labeling. The amounts of D-glucose- fect is due to partial aggregation of the protected incorporation of [3H]cytochalasin transporter, either with itself or other B were equivalent to 1.0,2.3, and 8.4 pmol/ polypeptides. mg protein applied to the gel for conditions i, ii, and iii, respectively. These values are Comparison with the Rat Adipocyte Transporter in the range of values (3 to 35 pmol/mg) found under slightly different conditions Previously, the same antiserum emduring the original photolabeling of the erythrocyte transporter and correspond to ployed in this study was used to detect the
204
KLIP ET AL.
photolabeling and immunological procedures to be the glucose transporter for the following reasons:
- 58 - 53 - 37 - 31
FIG. 3. The effect of sample preparation upon the autoradiogram pattern of the rat muscle plasma membranes. Samples were prepared for SDS-gel electrophoresis according to the procedure given under Experimental Procedures (no heating, lane A) or with the variation that the sample was held at 100°C for 2 min (lane B). Other conditions were as described in Fig. 2. The single nitrocellulose blot was treated with 160 fig of affinity-purified antibody.
glucose transporter in plasma membranes and microsomes from basal and insulinstimulated rat adipocytes (16). In that study, the samples for SDS-gel electrophoresis were heated at 100°C for 2 min, and the adipocyte transporter was identified in all the fractions as a 45,000-Da polypeptide. In order to compare the transporters from the two rat tissues directly, we have run heated and unheated SDS samples of plasma membranes from rat muscle and fat cells in a single gel. The mobilities of the transporters from the two tissues were identical after both methods of sample preparation (data not shown); thus the apparent molecular weight of the adipocyte transporter is also greater when the sample is not heated. DISCUSSION
We consider the polypeptide of molecular weight 45,000 to 50,000 identified by the
(i) The polypeptide is more extensively photolabeled by [3H]cytochalasin B in the presence of L- than D-ghCOSe. D-Glucose competitively inhibits the binding of [3H]cytochalasin B to the purified glucose transporter from human erythrocytes, whereas L-glucose does not (21). Although actin, which has a molecular weight of about 45,000, is also known to be photolabeled with [3H]cytochalasin B, D-glucose does not inhibit this labeling (6, 7, 22). (ii) The putative transporter polypeptide reacts with rabbit antibody against the purified transporter from human erythrocytes but not with control rabbit IgG. It could be argued that this reaction is fortuitous. However, the identification is strengthened by the fact that the muscle polypeptide has the same electrophoretic mobility as that of the adipocyte transporter previously identified with the same antiserum. In the case of the adipocyte, the relative amounts of the transporter polypeptide detected in various membrane fractions (plasma membranes and microsomes from basal and insulin-treated cells) by the immunological method were found to be in approximate agreement with the relative amounts of transporter measured by the binding of cytochalasin B (15,16). (iii) The apparent molecular weight of the muscle polypeptide is virtually the same as that of the purified glucose transporter from human erythrocytes. After partial removal of its extensive carbohydrate with glycosidase, the erythrocyte polypeptide runs as a species of molecular weight 46,000 on SDS-gel electrophoresis when the sample is heated and the gel is 10% polyacrylamide (19). In the case of the erythrocyte transporter, it has been shown that the purified protein exhibits the transport function in a reconstituted membrane (22, 23), and thus appears to constitute the entire transport system. The migration of the muscle transporter upon SDS-gel electrophoresis, relative to the molecular weight standards, which were largely water-soluble proteins, was
IDENTIFICATION
OF THE
MUSCLE
found to be sensitive to both the electrophoretic conditions (Fig. 1) and the method of sample preparation (Fig. 3). The reasons for these changes in relative mobility are unknown, but since the transporter is an integral membrane protein, their occurrence is not unexpected. The apparent molecular weight of the partially deglycosylated erythrocyte transporter varies from 43,000 on 7.5% acrylamide gels to 50,000 on 12.5% gels (19). For the purposes of purifying the muscle transporter and investigating its regulation it would be advantageous to achieve complete photolabeling with rH]cytochalasin B. However, for reasons that are not known, in none of the cell types in which the transporter has been photolabeled has more than a few percent labeling been obtained. In this regard, it is worth noting that the amounts of incorporated label may therefore not provide an accurate measure of the relative amounts of transporter under various conditions. Nevertheless, the availability of two methods to identify the muscle transporter should facilitate investigation of the molecular bases of the various types of regulation. Note added in proo$ Recently, the number of transporter-specific cytochalasin B binding sites was determined by the centrifugation procedure of reference (16). The values found are about B-fold larger than those reported herein, which were determined by an ultrafiltration assay. Evidently, a large fraction of the cytochalasin B bound to the transporter is lost during the wash of the filter. ACKNOWLEDGMENTS This research was supported by grants from the Medical Research Council of Canada to A.K. and from the National Institutes of Health to G.E.L. (GM 22996 and AM 25336). A.K. is an M.R.C. Scholar. D.S. is supported by a fellowship from the Juvenile Diabetes Foundation (No. 82F084). We are grateful to Dr. S. Grinstein and Dr. M. P. Czech for helpful suggestions concerning the photolabeling procedure, and to S. A. Olson and Dr. M. J. Weber for preparing the antiserum.
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TRANSPORTER
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3. KLIP, A., LOGAN, W. J., ANDLI, G. (1982) B&him Biophys. Actu 682,265-280. 4. WARDZALA, L. J., AND JEANRENAUD, B. (1981) J. Bid Chzem 256, 7090-7093. 5. GARTWAITE, S. M., AND HOLLOSZY, J. 0. (1982) J. Biol Chem 257, 5008-5012. 6. CARTER-SU, C., PESSIN, J. E., MORA, R., GITOMER, W., AND CZECH, M. P. (1982) J. Biol Chem. 257, 54165425. 7. SHANAHAN, M. F. (1982) J. Biol Chxm. 257,72907293. 8. PESSIN, J. E., TILLOTSON, L. G., YAMADA, K., GITOMER, W., CARTER-SU, C., MORA, R., ISSELBACHER, K. J., AND CZECH, M. P. (1982) Proc Natl Acad Sci USA 79,2286-2290. 9. SHANAHAN, M. F., OLSON, S. A., WEBER, M. J., LIENHARD, G. E., AND GORGA, J. C. (1982) Biochem. Biophys. Res. Ccnnmun 107,38-43. 10. PESSIN, J. E., GITOMER, W., AND CZECH, M. P. (1982) Dinbeti 31,29A. 11. JOHNSON, L. W., AND SMITH, C. H. (1982) Biochem. Biophys. Res. Commun. 109, 408-413. 12. BALDWIN, S. A., AND LIENHARD, G. E. (1980) Biochem Bisphys. Res Commun 94.1401-1408. 13. SOGIN, D. C., AND HINKLE, P. C. (1980) Proc. Natl. Acad Sci USA 77, 5725-5729. 14. SALTER, D. W., BALDWIN, S. A., LIENHARD, G. E., AND WEBER, M. J. (1982) Proc. Natl. Acad Sci USA 79, X40-1544. 15. WHEELER, T. J., SIMPSON, I. A., SOGIN, D. C., HINKLE, P. C., AND CUSHMAN, S. W. (1982) Biochem. Biophys. Res. Commun 105,89-95. 16. LIENHARD, G. E., KIM, H. H., RANSOME, K. J., AND GORGA, J. C. (1982) Bioch.em. Biophys. Res. Commun 105, 1150-1156. 17. KLIP, A., AND WALKER, D. (1983) Arch Biochem Biophys. 221, 175-187. 18. LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. L., AND RANDALL, R. J. (1951) J. Biol &em. 193, 265-275. 19. BALDWIN, S. A., BALDWIN, J. M., AND LIENHARD, G. E. (1982) Biochemistry 21.3836-3842. 20. LAEMMLI, U. K. (1970) Nature (Lo&on) 227,681685. 21. BALDWIN, S. A., BALDWIN, J. M., GORGA, F. R., AND LIENHARD, J. E. (1979) B&him Babphys. Ada 552, X33-188. 22. BALDWIN, J. M., GORGA, J. C., AND LIENHARD, G. E. (1981) J. Biol Chem 256,3685-3689. 23. WHEELER, T. J., AND HINKLE, P. C. (1981) J. Biol Owm. 256,8907-8914. 24. STECK, T. L. (1974) J. Cell Biol 62, 1-19. 25. WEBER, K., AND OSBORNE, M. (1975) in The Proteins (Neurath, H., and Hill, R. L., eds.), Vol. 1, pp. 179-223, Academic Press, New York. 26. AMES, GF.-L., ANDNIKAIDO, K. (1975) Bio&emist~ 15. 616-623.