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Glucose Deprivation Does Not Affect GLUT1 Targeting in 3T3-L1 Adipocytes
Biochemical and Biophysical Research Communications 273, 859 – 864 (2000) doi:10.1006/bbrc.2000.2985, available online at http://www.idealibrary.com on
Glucose Deprivation Does Not Affect GLUT1 Targeting in 3T3-L1 Adipocytes Robert J. McMahon, 1 Joseph B. Hwang, 2 and Susan C. Frost 3 Department of Biochemistry and Molecular Biology, University of Florida, Gainesville, Florida 32610
Received May 23, 2000
We have previously demonstrated that glucose deprivation alters the glycosylation of the GLUT1 glucose transporter in 3T3-L1 adipocytes. Many aberrantly glycosylated proteins are retained in the endoplasmic reticulum by interaction with chaperones. Herein, we use three independent procedures to show that GLUT1 is targeted to the plasma membrane, despite alterations in glycosylation. While earlier experiments revealed that plasma membrane targeting of aglyco GLUT 1 transporter was significantly reduced, our data show for the first time that altered glycosylation provides sufficient information to drive appropriate trafficking. © 2000 Academic Press Key Words: GLUT1; 3T3-L1 adipocytes; targeting; biotinylation; subcellular fractionation; plasma membrane fragments.
The GLUT1 glucose transporter is an integral membrane glycoprotein expressed in nearly all cells and is responsible for constitutive glucose uptake (1). The functional compartment of the GLUT1 is the plasma membrane, where it facilitates uptake of glucose into the cell. In 3T3-L1 adipocytes, GLUT1 is distributed both on the cell surface and intracellularly (2). We have shown that approximately 20% of GLUT1 resides in the plasma membrane in 3T3-L1 adipocytes based on data from recent subfractionation experiments (3). The remainder is ‘stored’ in an intracellular, vesicular compartment from which it can be recruited in the presence of hormone (2, 4 –7). The signals which target GLUT1 in the basal state to these two compartments and maintain the distribution between them are complex. Both the primary sequence and the glycosylation state may contribute to this targeting. By using chi1 Present address: Department of Food Science and Human Nutrition, Box 110370, University of Florida, Gainesville, FL 32611. 2 Present address: Department of Physiology, University of Michigan, School of Medicine, Ann Arbor, MI 48109. 3 To whom correspondence should be addressed. Fax: (904) 3922953. E-mail: [email protected].
meric constructs of GLUT1 and GLUT4 in Chinese Hamster Ovary (CHO) cells, Asano et al. (8) showed that domains within the interior of GLUT1 (from transmembrane spanning region 2 to 8) were important for plasma membrane targeting. However, Marshall et al. (9) showed that if the segment from amino acid residue 116 to 272, or 272 to 360, was replaced by the corresponding GLUT4 sequences that plasma membrane targeting in Xenopus oocytes was similar to that of wild-type GLUT1. Perhaps this suggests that structure as well as primary sequence is an important factor in targeting. GLUT1 contains an N-linked glycosylation consensus sequence (10) in the first exofacial loop. As a result of heterogeneous glycosylation at this site, the transporter migrates as a broad band on SDS–PAGE gels. Feugeas et al. showed that endoglycosidase treatment of reconstituted erythrocyte membranes reduced glucose transport activity (11). Likewise, treatment of chick embryo fibroblasts with the N-linked glycosylation inhibitor, tunicamycin, decreased glucose transport activity (12). By immunofluorescence staining, Asano et al. (13) showed that mutated GLUT1, in which either aspartate, tyrosine, or glutamine was replaced for the asparagine as position 45, was targeted to intracellular vesicles when expressed in CHO cells, rather than the plasma membrane. This supports their earlier study in which they demonstrated a reduction in transport activity in CHO cells transfected with these mutant clones compared to wild type GLUT1 (14). Together these data suggest that glycosylation is necessary for both activity and targeting. Left unanswered, however, is the question regarding abnormal glycosylation and its affect on targeting or activity. Glucose deprivation alters the glycosylation pattern of GLUT1 resulting in a low molecular weight species of the transporter. In some glucose-deprived cells, including 3T3-C2 fibroblasts and normal rat kidney cells, GLUT1 appears to lack oligosaccharide completely, based on insensitivity to endoglycosidase action (15, 16). In other cell types, including 3T3-L1 adipocytes (17–19) and CHO cells (20), the transporter is co-
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translationally glycosylated with a small core oligosaccharide, which is not further modified (21). This is consistent with earlier experiments which demonstrated that glucose deprivation leads to the synthesis of dolichol-linked Glc 3Man 5GlcNAc 2 rather than the normal Glc 3Man 9GlcNAc 2 to (22–28) N-linked glycoproteins containing this “alternative” structure are resistant to endoglycosidase H (endo H) in contrast to proteins which contain the normal core structure, which is the case with the lower molecular weight form of GLUT1 (21). This contrasts to GLUT1 produced in LEC1 CHO cells (21), a cell line which lacks N-acetylglucosaminyl transferase I activity in the Golgi (29). This deficiency prevents the synthesis of complex oligosaccharides, although the original structure derives from the normal, Glc 3Man 9GlcNAc 2 core. In this report, we have used the glucose deprivation model to determine if GLUT1 containing an alternative oligosaccharide structure targets to the plasma membrane. Using three entirely different technical approaches, we show that the lower molecular form of GLUT1 (p37) is targeted to the plasma membrane as efficiently as is the normal glycoform (p46) in 3T3-L1 adipocytes. Thus, p37 is targeted to the functional compartment despite the aberrant glycosylation. MATERIALS AND METHODS Materials Dulbecco’s Modified Eagle’s Medium (DMEM, high glucose) and DMEM base powder for the preparation of glucose-free medium was obtained from Life Technologies, Inc. Fetal bovine serum (#1020-75) and calf serum (#1100-90) were obtained from Intergen. Glucose-free fetal bovine serum was prepared by dialyzing against phosphatebuffered saline, pH 7.4, (PBS) for 48 h at 4°C, with a molecular weight cutoff of 13,000 (SpectraPor). Streptavidin-agarose, and sulfosuccinimidyl-6-(biotinamido) hexanoate (NHS-LC-Biotin) were obtained from Pierce. Poly-L-lysine (⬎100,000 mw) was from Sigma. Enhanced Chemiluminescent reagents were obtained from Amersham. The GLUT1 and GLUT4 rabbit polyclonal antibodies were generated against the respective C-terminal, thirteen amino acids. The GRP78 rabbit polyclonal antibody was generated against the first twelve amino acids in the N-terminus of the protein. The steel block homogenizer was purchased from Auburn Tool and Die (Warwick, RI). All other reagents were obtained at the highest quality commercially available.
Methods Cell culture. The culture and differentiation of 3T3-L1 cells was performed as described previously (30). Ten cm tissue culture plates, containing approximately 10 ⫻ 10 6 cells, were used for all experiments. For the plasma membrane lawn technique, cells were grown and differentiated on 22 mm glass coverslips. To glucose-deprive 3T3-L1 adipocytes, the normal culture medium was aspirated and replaced with glucose-free DMEM containing dialyzed fetal bovine serum. Subfractionation of 3T3-L1 adipocytes. Plasma membrane (PM), high density (HDM), and low density membrane (LDM) fractions were isolated by a technique developed by Fisher and Frost (31), which is a modification of one described previously (31). Briefly,
control or glucose-deprived 3T3-L1 adipocytes were scraped into TES (10 mM Tris-HCl, pH 7.4, 1 mM EDTA, and 250 mM sucrose) at 18°C. The cells were passed over a tungsten ball ten times in a steel block homogenizer (at 4°C) which provided a clearance of 0.0025 in. A crude plasma membrane fraction was collected at 17,369g for 15 min at 4°C. Purified membranes were collected from this fraction by sucrose gradient centrifugation. HDM and LDM fractions were collected by differential centrifugation. Membrane fractions were stored in TES at ⫺20°C. Protein was determined by the method of Markwell et al. (32). For protein separation, samples were diluted into sample dilution buffer containing 6M urea and 10% -mercaptoethanol before separation by SDS–PAGE and transfer to nitrocellulose as described previously (19). Immunoblot analysis of GLUT1, GLUT4, and GRP78 was accomplished using the ECL system as previously described (19). Cell surface biotinylation and recovery of biotinylation membrane proteins. The procedure used for the biotinylation of 3T3-L1 adipocyte plasma membranes was modified from a previously described method (33). 3T3-L1 adipocytes were rinsed at 4°C in PBS, pH, 7.4. The cells were then incubated in PBS, pH 8.5 in the presence or absence of NHS-LC-Biotin (0.5 mg/ml) for 1 h at 4°C. The solution was removed and the reaction quenched by addition of TES. The cells were then scraped into 3.5 ml TES (containing protease inhibitors: aprotinin, leupeptin, pepstatin, TPCK, and TLCK at 2 g/ml, and 1 mM PMSF) and sonicated for 10 s on power 2 at 50% duty cycle (Branson Sonifier 450). A total membrane fraction was collected by centrifugation at 212,000g for 1 h at 4°C. The entire pellet was resuspended in PES extraction buffer (PBS containing 1 mM EDTA, protease inhibitors as above, 0.1% SDS, and 2% polyoxyethylene 9 lauryl ether) and sonicated. Insoluble material was then removed by centrifugation at 13,300 ⫻ g for 5 min at 4°C. The clarified supernatant was then incubated with 50 L streptavidin-agarose for 6 h at 4°C with end-over-end rotation. The agarose beads was collected by brief centrifugation in a microcentrifuge and washed five times in extraction buffer (three times for 1 min each and 2 times for 10 min each). Specifically bound proteins were released by incubation in sample dilution buffer containing 6M urea and 10% -mercaptoethanol at 37°C for 30 min. Released proteins were resolved by 7.5% SDS–PAGE. Detection of biotinylated proteins and GLUT1 transporter. Resolved proteins were transferred to nitrocellulose in the buffer described previously (19). Nonspecific binding was blocked in TBS-T (20 mM Tris-base, pH 7.5, 150 mM NaCl, 0.1% Tween-20) containing 5% nonfat dry milk (Carnation) for 30 min at room temperature. For the detection of total biotinylated proteins, blots were washed in TBS-T and then incubated in TBS-T containing 0.5% nonfat dry milk and 2 ⫻ 10 ⫺5 mg/ml streptavidin-horseradish peroxidase conjugate for 1 h. The blot was then washed extensively in TBS-T and then visualized by incubation in ECL reagents and exposure to film (ranging from 10 s to 10 min). For the detection of biotinylated GLUT1, blots were incubated in TBS-T containing 5% NFDM and 1:500 dilution of anti-GLUT1 serum for 1 h at room temperature. The blot was then washed in TBS-T before being incubated in TBS-T containing 5% NFDM and 1:50,000 dilution of goat anti-rabbit IgGhorseradish peroxidase conjugate for 1 h at room temperature. The blot was then visualized by chemiluminescence. Quantitation was performed by video densitometry on a Visage Bioscan in the linear range of the film and detection system. Plasma membrane fragment isolation. Cells, adherent to 22 mm glass coverslips, were washed in PBS at 4°C. The glass coverslips were placed in clean 35 mm tissue culture dishes before incubating in 1 mL PBS containing 0.5 mg poly-L-lysine (M.W. ⬎100,000) for 1 min. The monolayer was then washed three times at 4°C with 3.0 mL sonication buffer (70 mM KCl, 30 mM HEPES, pH 7.5, 5 mM MgCl 2, and 3 mM EGTA) diluted 1:3 with dH 2O. This solution was aspirated and replaced with 1 mL sonication buffer at 4°C. The monolayer was the immediately placed under a 21-inch tapped flat horn at a distance
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FIG. 1. Identification of GLUT1 in isolated membrane fractions. Cells were incubated in the presence (F) or absence (S) of 25 mM glucose for 48 h before subcellular fractionation as described under Materials and Methods. Membrane proteins (50 g/lane) were resolved by 7.5% SDS–PAGE and probed for GRP78, GLUT4, and GLUt1 after electrophoretic transfer to nitrocellulose. The result shown is representative of at least two independent experiments.
of 5 mm and sonicated for approximately 2 s at power of 1.5 on a Heat Systems Sonicator. The glass coverslips were then washed three times at 4°C in PBS. All traces of PBS were carefully removed from the coverslip before addition of 50 L of sample dilution buffer. The lysed cells were scraped from the cover slip and loaded onto a mini SDS–PAGE gel for protein separation. The presence of GLUT1 and GRP78 were detected using appropriate antibodies.
RESULTS AND DISCUSSION Subfractionation of Control and Glucose-Deprived 3T3-L1 Adipocytes To determine if truncated glycosylation permits GLUT1 targeting to the cell surface, we first took advantage of a subfractionation technique, recently developed in our laboratory, to isolate plasma membranes from intracellular vesicles all of which contain GLUT1 (3). By this technique, we have shown that about 20% of GLUT1 resides in the plasma membrane, a fraction which contains a 12-fold enrichment of cellsurface proteins. Figure 1 compares the expression of GLUT1 in membranes from control (F) to those from glucose-deprived (S) adipocytes. The normal glycoform, migrating as a 46 kDa species (p46), was detected in each fraction of control and glucose-deprived cells. In membranes isolated from glucose-deprived cells, the lower molecular weight glycoform of GLUT1, which migrates as a 37 kDa protein (p37), as well as p46, was identified. The ratio of p46 to p37 in the plasma membrane (2.1) was similar to that in the homogenate (2.4). This suggests that the efficiency of targeting of p37 and p46 is similar, as the PM compartment reflects the total pool (homogenate). One potential problem with subfractionation procedures is the potential “contamination” of one membrane fraction with proteins from another membrane fraction. To address this possibility, we have probed the various fractions for GLUT4,
the insulin-sensitive glucose transporter, which is localized primarily to the endosomal compartment in the absence of insulin (34), and GRP78, an endoplasmic reticulum (ER) chaperone (35). In control cells, GRP78 migrated primarily with the HDM fraction, although a band was clearly visible in the PM fraction as well. Based on the densitometric analysis of these bands, 12% of the GRP78 pool migrated with the PM fraction. This suggests that 12% of the ER is associated with the PM. Whether this represents cross contamination is explored in more detail below (see Fig. 3). Glucose deprivation increased the expression of GRP78 expression, as expected from previous work (36), seen in each fraction as well as the homogenate. However, the percent comigration of this ER marker with the PM was the same as in controls. GLUT4 in control cells migrated in both the LDM and HDM fractions, although there was some in the PM fraction as well (approximately 3% of the total). In the glucose-deprived cells, the expression of GLUT4 in the LDM and HDM fractions was reduced, consistent with the loss of GLUT4 mRNA as reported previously (18, 19). No redistribution to the PM was noted with glucose deprivation, confirming our earlier study (3). Cell Surface Biotinylation of Control and Glucose-Deprived 3T3-L1 Adipocytes The potential contamination of PM with intracellular membrane proteins by the fractionation procedure confounds data interpretation. Thus, we sought alternative methods for determining the localization of p37. Modification of cell-surface proteins with membraneimpermeant biotinylation reagents has proven to be an effective tool for examining protein trafficking of the vitronectin receptor (37), Thy-1 and lymphocyte surface marker proteins (38), surface protein of both 3T3-L1 fibroblasts (33, 39) and 3T3-L1 adipocytes (33), insulin receptors (40), and tumor necrosis factor receptor (41). To first demonstrate the overall pattern of biotinylated cell surface membrane proteins, adipocytes were fed or glucose-deprived for 48 h to allow sufficient accumulation of p37 (in the deprived condition). Subsequent to biotinylation, membranes were extracted and biotinylated proteins recovered with strepavidin-agarose. The released proteins were resolved by SDS–PAGE, transferred to nitrocellulose, and probed for biotinylated proteins using strepavidinlinked horse radish peroxidase (Fig. 2). In cells which were not biotinylated, two bands were detected, corresponding to proteins of 120 kDa and 68 kDa (A, ⫺ biotinylation). These likely represent proteins with endogenous biotin, perhaps entrapped carboxylases which represent a large proportion of soluble proteins in 3T3-L1 adipocytes (42, 43). In cells modified with the membrane impermeant biotinylation reagent, a number of proteins were observed (A, ⫹ biotinylation).
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FIG. 2. Cell surface biotinylation and detection of GLUT1. Cells were incubated in the presence or absence of 25 mM glucose for 48 h before biotinylation with NHS-LC biotin as described under Materials and Methods. Biotinylated proteins were recovered with strepavidin-agarose and resolved by 7.5% SDS–PAGE. (A) Biotinylated proteins, released from the streptavidin-agarose, were detected by a modified immunoblotting technique using strepavidin-HRP. Molecular weight markers are shown to the left of the panel. (B) GLUT1, among the biotinylated protein pool, was detected by immunoblot analysis. The positions of p46 and p37 are indicated by the arrows, along with molecular weight markers. These results are representative of at least three independent experiments.
Careful comparison between the glucose-fed and glucose-deprived cells revealed distinct differences, which represent proteins present at the cell surface in one metabolic state and not the other. A duplicate experiment was performed for the immunological identification of GLUT1, using a GLUT1-specific antibody, amongst the biotinylated proteins captured with strepavidin agarose (B). As expected, the normal GLUT1 glycoform was detected at the cell surface of fed cells (B, ⫹ biotinylation). Note that in cells which were not biotinylated, no GLUT1 could be detected (B, ⫺ biotinylation). In samples from glucose-deprived cells, both p46 and p37 were detected. The ratio of p46:p37 was three, slightly higher than that in the PM fraction isolated by the subfractionation procedure.
coverslip surface with polylysine, and sonicated. Recovery of GLUT1 and GRP78 in these membrane fragments was analyzed by SDS–PAGE/immunoblot analysis in comparison to total membranes (Fig. 3). As expected, p46 and GRP78 were detected in total membranes isolated from control cells. In total membranes from glucose-deprived cells, both p37 and p46 were detected along with induced expression of GRP78, as noted in Fig. 1. Plasma membrane fragments from control cells contained p46, but interestingly, no GRP78. Plasma membrane fragments from glucosedeprived cells contained both p46 and p37, but again no GRP78, despite its induced expression. This demonstrates that the plasma membrane fragments were free of intracellular membranes and yet p37 was still observed in fragments from the glucose-deprived cells. This further indicates that the comigration of GRP78 in the subfractionation procedure represents contamination with ER membranes. The ratio of p46:p37 in total membranes was similar to that in PM fragments (2.2 vs 2.5). The data presented herein shows for the first time that GLUT1, when alternatively glycosylated, is targeted in similar if not identical fashion to the normal GLUT1 glycoform. In fact, the similarity in the ratio of these two forms in the PM fraction relative to the homogenate or total membrane fragments, also suggests that the recycling rates of the p46 and p37 (i.e., the movement from the intracellular storage sites to the PM and back again) are the same. In contrast, others have shown that aglyco GLUT1 (i.e., mutagenized transporter lacking the N-linked glycosylation site) is retained within intracellular vesicles (13). This suggests that the presence of oligosaccharide, even though abnormal, is sufficient to provide the appropriate targeting and recycling information. This appears to be the case as well in the LEC1 CHO cells (48)
Plasma Membrane Fragment Isolation The isolation of plasma membrane fragments has been used for characterizing the assembly of clathrincoated pits which occurs at the inner face of the plasma membrane (44, 45) and the purification of caveolin (46). Recently, the technique has been modified for the image analysis of GLUT4 recruitment to the plasma membrane in response to insulin treatment (47). We therefore undertook this method to generate plasma membrane fragments which are potentially free from contamination of endoplasmic reticulum and Golgi apparatus. Adipocytes grown on glass coverslips were deprived of glucose (or not) for 48 h, cross-linked to the
FIG. 3. Identification of GLUT1 in plasma membrane fragments. Cells were incubated in the presence or absence of 25 mM glucose for 48 h before plasma membrane fragments were isolated as described under Materials and Methods. Proteins in the total membrane samples, along with plasma membrane fragments, were separated by SDS–PAGE and probed for GRP78 and GLUT1 after electrophoretic transfer. These data are representative of three independent experiments.
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which lack N-acetylglucosaminyl transferase I (29) resulting in an abbreviated but normal core structure. The functional status of p37 is unknown at this point because of the technical difficulties in separating p37containing membranes from p46-containing membranes. However, we can speculate that p37 transports glucose with less efficiency than p46 from the following observations. Glucose transport activity increases upon glucose deprivation (19). At 24 h of deprivation, the difference can be as much as 10- to 15-fold higher than controls. However, between 24 to 48 h of deprivation, this activity begins to decay . . . such that at 48 h, almost 40% of the “activated” transport activity is lost. P37 appears at about 18 h becoming an increasingly significant percent of the total pool as time progresses. The accumulation of p37 during extended glucose deprivation (24 to 48 h) is inversely correlated with the increase in transport activity. Secondly, glucosedeprived cells supplemented with fructose, exhibit the same “activated” transport activity as cells deprived of glucose in the absence of fructose (19). However, the appearance and accumulation of p37 is completely prevented. This is likely because fructose supplies the precursors for both N-acetylglucosamine and GDP mannose (both of which are important in core oligosaccharide synthesis). Importantly, the transport activity does not decay after 24 h of deprivation. Together these data argue that p37 has less intrinsic activity than does p46, despite the normal targeting. REFERENCES 1. Bell, G. I., Burant, C. F., Takeda, J., and Gould, G. W. (1993) Structure and function of mammalian facilitative sugar transporters. J. Biol. Chem. 268, 19161–19164. 2. Piper, R. C., Hess, L. J., and James, D. E. (1991) Differential sorting of two glucose transporters expressed in insulin-sensitive cells. Am. J. Physiol. 260, C570 –C580. 3. Fisher, M. D., and Frost, S. C. (1996) Translocation of GLUT1 does not account for elevated glucose transport in glucosedeprived 3T3-L1 adipocytes. J. Biol. Chem. 271, 11806 –11809. 4. Calderhead, D. M., Kitagawa, K., Tanner, L. I., Holman, G. D., and Lienhard, G. E. (1990) Insulin regulation of the two glucose transporters in 3T3-L1 adipocytes. J. Biol. Chem. 265, 13800 – 13808. 5. Satoh, S., Nishimura, H., Clark, A. E., Kozka, I. J., Vannucci, S. J., Simpson, I. A., Quon, M. J., Cushman, S. W., and Holman, G. D. (1993) Use of bismannose photolabel to elucidate insulinregulated GLUT4 subcellular trafficking kinetics in rat adipose cells. J. Biol. Chem. 268, 17820 –17829. 6. Yang, J., and Holman, G. D. (1993) Comparison of GLUT 4 and Glut 1 subcellular trafficking in basal and insulin-stimulated 3T3-L1 cells. J. Biol. Chem. 268, 4600 – 4603. 7. Robinson, L. J., and James, D. E. (1992) Insulin-regulated sorting of glucose transporters in 3T3-L1 adipocytes. Am. J. Physiol. 263, E383–E393. 8. Asano, T., Takata, K., Katagiri, H., Tsukuda, K., Lin, J.-L., Ishihara, H., Inukai, K., Hirano, H., Yazaki, Y., and Oka, Y. (1992) Domains responsible for the differential targeting of glucose transporter isoforms. J. Biol. Chem. 267, 19636 –19641.
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D. (1992) Immunodetection of biotinylated lymphocyte-surface proteins by enhanced chemiluminescence: A nonradioactive method for cell-surface protein analysis. Anal. Biochem. 204, 220 –226. Hare, J. F., and Taylor, K. (1991) Mechanisms of plasma membrane protein degradation: Recycling proteins are degraded more rapidly than those confined to the cell surface. Proc. Natl. Acad. Sci. USA 88, 5902–5906. Levy-Toledano, R., Heleen, L., Caro, P., Hindman, N., and Taylor, S. (1993) Streptavidin blotting: A sensitive technique to study cell surface proteins; Application to investigate autophosphorylation and endocytosis of biotin-labeled insulin receptors. Endocrinology 133, 1803–1808. Hsu, K. C., and Chao, M. V. (1993) Differential expression and ligand binding properties of tumor necrosis factor receptor chimeric mutants. J. Biol. Chem. 268, 16430 –16436. Mackall, J. C., Student, A. K., Polakis, E. S., and Lane, M. D. (1976) Induction of lipogenesis during differentiation in a “preadipocyte” cell line. J. Biol. Chem. 251, 6462– 6464. Mackall, J. C., and Lane, M. D. (1977) Role of pyruvate carboxylase in fatty acid synthesis: Alterations during preadipocyte differentiation. Biochem. Biophys. Res. Comm. 79, 720 –725. Lin, H. C., Moore, M. S., Sanan, D. A., and Anderson, R. G. (1991) Reconstitution of clathrin-coated pit budding from plasma membranes. J. Cell Biol. 114, 881– 891. Lin, H. C., Sudhof, T. C., and Anderson, R. G. (1992) Annexin VI is required for budding of clathrin-coated pits. Cell 70, 283–291. Chang, W. J., Ying, Y. S., Rothberg, K. G., Hooper, N. M., Turner, A. J., Gambliel, H. A., De-Gunzberg, J., Mumby, S. M., Gilman, A. G., and Anderson, R. G. (1994) Purification and characterization of smooth muscle cell caveolae. J. Cell Biol. 126, 127–138. Robinson, L. J., Pang, S., Harris, D. S., Heuser, J., and James, D. E. (1992) Translocation of the glucose transporter (GLUT4) to the surface in permeabilized 3T3-L1 adipocytes: Effect of ATP, insulin, and GTPgammaS and localization of GLUT4 to clathrin lattices. J. Cell Biol. 117, 1181–1196. Haspel, H. C., Revillame, J., and Rosen, O. M. (1988) Structure, biosynthesis, and function of the hexose transporter in chinese hamster ovary cells deficient in N-acetylglucosaminyltransferase 1 activity. J. Cell. Physiol. 136, 361–366.
Glucose Deprivation Does Not Affect GLUT1 Targeting in 3T3-L1 Adipocytes Robert J. McMahon, 1 Joseph B. Hwang, 2 and Susan C. Frost 3 Department of Biochemistry and Molecular Biology, University of Florida, Gainesville, Florida 32610
Received May 23, 2000
We have previously demonstrated that glucose deprivation alters the glycosylation of the GLUT1 glucose transporter in 3T3-L1 adipocytes. Many aberrantly glycosylated proteins are retained in the endoplasmic reticulum by interaction with chaperones. Herein, we use three independent procedures to show that GLUT1 is targeted to the plasma membrane, despite alterations in glycosylation. While earlier experiments revealed that plasma membrane targeting of aglyco GLUT 1 transporter was significantly reduced, our data show for the first time that altered glycosylation provides sufficient information to drive appropriate trafficking. © 2000 Academic Press Key Words: GLUT1; 3T3-L1 adipocytes; targeting; biotinylation; subcellular fractionation; plasma membrane fragments.
The GLUT1 glucose transporter is an integral membrane glycoprotein expressed in nearly all cells and is responsible for constitutive glucose uptake (1). The functional compartment of the GLUT1 is the plasma membrane, where it facilitates uptake of glucose into the cell. In 3T3-L1 adipocytes, GLUT1 is distributed both on the cell surface and intracellularly (2). We have shown that approximately 20% of GLUT1 resides in the plasma membrane in 3T3-L1 adipocytes based on data from recent subfractionation experiments (3). The remainder is ‘stored’ in an intracellular, vesicular compartment from which it can be recruited in the presence of hormone (2, 4 –7). The signals which target GLUT1 in the basal state to these two compartments and maintain the distribution between them are complex. Both the primary sequence and the glycosylation state may contribute to this targeting. By using chi1 Present address: Department of Food Science and Human Nutrition, Box 110370, University of Florida, Gainesville, FL 32611. 2 Present address: Department of Physiology, University of Michigan, School of Medicine, Ann Arbor, MI 48109. 3 To whom correspondence should be addressed. Fax: (904) 3922953. E-mail: [email protected].
meric constructs of GLUT1 and GLUT4 in Chinese Hamster Ovary (CHO) cells, Asano et al. (8) showed that domains within the interior of GLUT1 (from transmembrane spanning region 2 to 8) were important for plasma membrane targeting. However, Marshall et al. (9) showed that if the segment from amino acid residue 116 to 272, or 272 to 360, was replaced by the corresponding GLUT4 sequences that plasma membrane targeting in Xenopus oocytes was similar to that of wild-type GLUT1. Perhaps this suggests that structure as well as primary sequence is an important factor in targeting. GLUT1 contains an N-linked glycosylation consensus sequence (10) in the first exofacial loop. As a result of heterogeneous glycosylation at this site, the transporter migrates as a broad band on SDS–PAGE gels. Feugeas et al. showed that endoglycosidase treatment of reconstituted erythrocyte membranes reduced glucose transport activity (11). Likewise, treatment of chick embryo fibroblasts with the N-linked glycosylation inhibitor, tunicamycin, decreased glucose transport activity (12). By immunofluorescence staining, Asano et al. (13) showed that mutated GLUT1, in which either aspartate, tyrosine, or glutamine was replaced for the asparagine as position 45, was targeted to intracellular vesicles when expressed in CHO cells, rather than the plasma membrane. This supports their earlier study in which they demonstrated a reduction in transport activity in CHO cells transfected with these mutant clones compared to wild type GLUT1 (14). Together these data suggest that glycosylation is necessary for both activity and targeting. Left unanswered, however, is the question regarding abnormal glycosylation and its affect on targeting or activity. Glucose deprivation alters the glycosylation pattern of GLUT1 resulting in a low molecular weight species of the transporter. In some glucose-deprived cells, including 3T3-C2 fibroblasts and normal rat kidney cells, GLUT1 appears to lack oligosaccharide completely, based on insensitivity to endoglycosidase action (15, 16). In other cell types, including 3T3-L1 adipocytes (17–19) and CHO cells (20), the transporter is co-
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translationally glycosylated with a small core oligosaccharide, which is not further modified (21). This is consistent with earlier experiments which demonstrated that glucose deprivation leads to the synthesis of dolichol-linked Glc 3Man 5GlcNAc 2 rather than the normal Glc 3Man 9GlcNAc 2 to (22–28) N-linked glycoproteins containing this “alternative” structure are resistant to endoglycosidase H (endo H) in contrast to proteins which contain the normal core structure, which is the case with the lower molecular weight form of GLUT1 (21). This contrasts to GLUT1 produced in LEC1 CHO cells (21), a cell line which lacks N-acetylglucosaminyl transferase I activity in the Golgi (29). This deficiency prevents the synthesis of complex oligosaccharides, although the original structure derives from the normal, Glc 3Man 9GlcNAc 2 core. In this report, we have used the glucose deprivation model to determine if GLUT1 containing an alternative oligosaccharide structure targets to the plasma membrane. Using three entirely different technical approaches, we show that the lower molecular form of GLUT1 (p37) is targeted to the plasma membrane as efficiently as is the normal glycoform (p46) in 3T3-L1 adipocytes. Thus, p37 is targeted to the functional compartment despite the aberrant glycosylation. MATERIALS AND METHODS Materials Dulbecco’s Modified Eagle’s Medium (DMEM, high glucose) and DMEM base powder for the preparation of glucose-free medium was obtained from Life Technologies, Inc. Fetal bovine serum (#1020-75) and calf serum (#1100-90) were obtained from Intergen. Glucose-free fetal bovine serum was prepared by dialyzing against phosphatebuffered saline, pH 7.4, (PBS) for 48 h at 4°C, with a molecular weight cutoff of 13,000 (SpectraPor). Streptavidin-agarose, and sulfosuccinimidyl-6-(biotinamido) hexanoate (NHS-LC-Biotin) were obtained from Pierce. Poly-L-lysine (⬎100,000 mw) was from Sigma. Enhanced Chemiluminescent reagents were obtained from Amersham. The GLUT1 and GLUT4 rabbit polyclonal antibodies were generated against the respective C-terminal, thirteen amino acids. The GRP78 rabbit polyclonal antibody was generated against the first twelve amino acids in the N-terminus of the protein. The steel block homogenizer was purchased from Auburn Tool and Die (Warwick, RI). All other reagents were obtained at the highest quality commercially available.
Methods Cell culture. The culture and differentiation of 3T3-L1 cells was performed as described previously (30). Ten cm tissue culture plates, containing approximately 10 ⫻ 10 6 cells, were used for all experiments. For the plasma membrane lawn technique, cells were grown and differentiated on 22 mm glass coverslips. To glucose-deprive 3T3-L1 adipocytes, the normal culture medium was aspirated and replaced with glucose-free DMEM containing dialyzed fetal bovine serum. Subfractionation of 3T3-L1 adipocytes. Plasma membrane (PM), high density (HDM), and low density membrane (LDM) fractions were isolated by a technique developed by Fisher and Frost (31), which is a modification of one described previously (31). Briefly,
control or glucose-deprived 3T3-L1 adipocytes were scraped into TES (10 mM Tris-HCl, pH 7.4, 1 mM EDTA, and 250 mM sucrose) at 18°C. The cells were passed over a tungsten ball ten times in a steel block homogenizer (at 4°C) which provided a clearance of 0.0025 in. A crude plasma membrane fraction was collected at 17,369g for 15 min at 4°C. Purified membranes were collected from this fraction by sucrose gradient centrifugation. HDM and LDM fractions were collected by differential centrifugation. Membrane fractions were stored in TES at ⫺20°C. Protein was determined by the method of Markwell et al. (32). For protein separation, samples were diluted into sample dilution buffer containing 6M urea and 10% -mercaptoethanol before separation by SDS–PAGE and transfer to nitrocellulose as described previously (19). Immunoblot analysis of GLUT1, GLUT4, and GRP78 was accomplished using the ECL system as previously described (19). Cell surface biotinylation and recovery of biotinylation membrane proteins. The procedure used for the biotinylation of 3T3-L1 adipocyte plasma membranes was modified from a previously described method (33). 3T3-L1 adipocytes were rinsed at 4°C in PBS, pH, 7.4. The cells were then incubated in PBS, pH 8.5 in the presence or absence of NHS-LC-Biotin (0.5 mg/ml) for 1 h at 4°C. The solution was removed and the reaction quenched by addition of TES. The cells were then scraped into 3.5 ml TES (containing protease inhibitors: aprotinin, leupeptin, pepstatin, TPCK, and TLCK at 2 g/ml, and 1 mM PMSF) and sonicated for 10 s on power 2 at 50% duty cycle (Branson Sonifier 450). A total membrane fraction was collected by centrifugation at 212,000g for 1 h at 4°C. The entire pellet was resuspended in PES extraction buffer (PBS containing 1 mM EDTA, protease inhibitors as above, 0.1% SDS, and 2% polyoxyethylene 9 lauryl ether) and sonicated. Insoluble material was then removed by centrifugation at 13,300 ⫻ g for 5 min at 4°C. The clarified supernatant was then incubated with 50 L streptavidin-agarose for 6 h at 4°C with end-over-end rotation. The agarose beads was collected by brief centrifugation in a microcentrifuge and washed five times in extraction buffer (three times for 1 min each and 2 times for 10 min each). Specifically bound proteins were released by incubation in sample dilution buffer containing 6M urea and 10% -mercaptoethanol at 37°C for 30 min. Released proteins were resolved by 7.5% SDS–PAGE. Detection of biotinylated proteins and GLUT1 transporter. Resolved proteins were transferred to nitrocellulose in the buffer described previously (19). Nonspecific binding was blocked in TBS-T (20 mM Tris-base, pH 7.5, 150 mM NaCl, 0.1% Tween-20) containing 5% nonfat dry milk (Carnation) for 30 min at room temperature. For the detection of total biotinylated proteins, blots were washed in TBS-T and then incubated in TBS-T containing 0.5% nonfat dry milk and 2 ⫻ 10 ⫺5 mg/ml streptavidin-horseradish peroxidase conjugate for 1 h. The blot was then washed extensively in TBS-T and then visualized by incubation in ECL reagents and exposure to film (ranging from 10 s to 10 min). For the detection of biotinylated GLUT1, blots were incubated in TBS-T containing 5% NFDM and 1:500 dilution of anti-GLUT1 serum for 1 h at room temperature. The blot was then washed in TBS-T before being incubated in TBS-T containing 5% NFDM and 1:50,000 dilution of goat anti-rabbit IgGhorseradish peroxidase conjugate for 1 h at room temperature. The blot was then visualized by chemiluminescence. Quantitation was performed by video densitometry on a Visage Bioscan in the linear range of the film and detection system. Plasma membrane fragment isolation. Cells, adherent to 22 mm glass coverslips, were washed in PBS at 4°C. The glass coverslips were placed in clean 35 mm tissue culture dishes before incubating in 1 mL PBS containing 0.5 mg poly-L-lysine (M.W. ⬎100,000) for 1 min. The monolayer was then washed three times at 4°C with 3.0 mL sonication buffer (70 mM KCl, 30 mM HEPES, pH 7.5, 5 mM MgCl 2, and 3 mM EGTA) diluted 1:3 with dH 2O. This solution was aspirated and replaced with 1 mL sonication buffer at 4°C. The monolayer was the immediately placed under a 21-inch tapped flat horn at a distance
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FIG. 1. Identification of GLUT1 in isolated membrane fractions. Cells were incubated in the presence (F) or absence (S) of 25 mM glucose for 48 h before subcellular fractionation as described under Materials and Methods. Membrane proteins (50 g/lane) were resolved by 7.5% SDS–PAGE and probed for GRP78, GLUT4, and GLUt1 after electrophoretic transfer to nitrocellulose. The result shown is representative of at least two independent experiments.
of 5 mm and sonicated for approximately 2 s at power of 1.5 on a Heat Systems Sonicator. The glass coverslips were then washed three times at 4°C in PBS. All traces of PBS were carefully removed from the coverslip before addition of 50 L of sample dilution buffer. The lysed cells were scraped from the cover slip and loaded onto a mini SDS–PAGE gel for protein separation. The presence of GLUT1 and GRP78 were detected using appropriate antibodies.
RESULTS AND DISCUSSION Subfractionation of Control and Glucose-Deprived 3T3-L1 Adipocytes To determine if truncated glycosylation permits GLUT1 targeting to the cell surface, we first took advantage of a subfractionation technique, recently developed in our laboratory, to isolate plasma membranes from intracellular vesicles all of which contain GLUT1 (3). By this technique, we have shown that about 20% of GLUT1 resides in the plasma membrane, a fraction which contains a 12-fold enrichment of cellsurface proteins. Figure 1 compares the expression of GLUT1 in membranes from control (F) to those from glucose-deprived (S) adipocytes. The normal glycoform, migrating as a 46 kDa species (p46), was detected in each fraction of control and glucose-deprived cells. In membranes isolated from glucose-deprived cells, the lower molecular weight glycoform of GLUT1, which migrates as a 37 kDa protein (p37), as well as p46, was identified. The ratio of p46 to p37 in the plasma membrane (2.1) was similar to that in the homogenate (2.4). This suggests that the efficiency of targeting of p37 and p46 is similar, as the PM compartment reflects the total pool (homogenate). One potential problem with subfractionation procedures is the potential “contamination” of one membrane fraction with proteins from another membrane fraction. To address this possibility, we have probed the various fractions for GLUT4,
the insulin-sensitive glucose transporter, which is localized primarily to the endosomal compartment in the absence of insulin (34), and GRP78, an endoplasmic reticulum (ER) chaperone (35). In control cells, GRP78 migrated primarily with the HDM fraction, although a band was clearly visible in the PM fraction as well. Based on the densitometric analysis of these bands, 12% of the GRP78 pool migrated with the PM fraction. This suggests that 12% of the ER is associated with the PM. Whether this represents cross contamination is explored in more detail below (see Fig. 3). Glucose deprivation increased the expression of GRP78 expression, as expected from previous work (36), seen in each fraction as well as the homogenate. However, the percent comigration of this ER marker with the PM was the same as in controls. GLUT4 in control cells migrated in both the LDM and HDM fractions, although there was some in the PM fraction as well (approximately 3% of the total). In the glucose-deprived cells, the expression of GLUT4 in the LDM and HDM fractions was reduced, consistent with the loss of GLUT4 mRNA as reported previously (18, 19). No redistribution to the PM was noted with glucose deprivation, confirming our earlier study (3). Cell Surface Biotinylation of Control and Glucose-Deprived 3T3-L1 Adipocytes The potential contamination of PM with intracellular membrane proteins by the fractionation procedure confounds data interpretation. Thus, we sought alternative methods for determining the localization of p37. Modification of cell-surface proteins with membraneimpermeant biotinylation reagents has proven to be an effective tool for examining protein trafficking of the vitronectin receptor (37), Thy-1 and lymphocyte surface marker proteins (38), surface protein of both 3T3-L1 fibroblasts (33, 39) and 3T3-L1 adipocytes (33), insulin receptors (40), and tumor necrosis factor receptor (41). To first demonstrate the overall pattern of biotinylated cell surface membrane proteins, adipocytes were fed or glucose-deprived for 48 h to allow sufficient accumulation of p37 (in the deprived condition). Subsequent to biotinylation, membranes were extracted and biotinylated proteins recovered with strepavidin-agarose. The released proteins were resolved by SDS–PAGE, transferred to nitrocellulose, and probed for biotinylated proteins using strepavidinlinked horse radish peroxidase (Fig. 2). In cells which were not biotinylated, two bands were detected, corresponding to proteins of 120 kDa and 68 kDa (A, ⫺ biotinylation). These likely represent proteins with endogenous biotin, perhaps entrapped carboxylases which represent a large proportion of soluble proteins in 3T3-L1 adipocytes (42, 43). In cells modified with the membrane impermeant biotinylation reagent, a number of proteins were observed (A, ⫹ biotinylation).
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FIG. 2. Cell surface biotinylation and detection of GLUT1. Cells were incubated in the presence or absence of 25 mM glucose for 48 h before biotinylation with NHS-LC biotin as described under Materials and Methods. Biotinylated proteins were recovered with strepavidin-agarose and resolved by 7.5% SDS–PAGE. (A) Biotinylated proteins, released from the streptavidin-agarose, were detected by a modified immunoblotting technique using strepavidin-HRP. Molecular weight markers are shown to the left of the panel. (B) GLUT1, among the biotinylated protein pool, was detected by immunoblot analysis. The positions of p46 and p37 are indicated by the arrows, along with molecular weight markers. These results are representative of at least three independent experiments.
Careful comparison between the glucose-fed and glucose-deprived cells revealed distinct differences, which represent proteins present at the cell surface in one metabolic state and not the other. A duplicate experiment was performed for the immunological identification of GLUT1, using a GLUT1-specific antibody, amongst the biotinylated proteins captured with strepavidin agarose (B). As expected, the normal GLUT1 glycoform was detected at the cell surface of fed cells (B, ⫹ biotinylation). Note that in cells which were not biotinylated, no GLUT1 could be detected (B, ⫺ biotinylation). In samples from glucose-deprived cells, both p46 and p37 were detected. The ratio of p46:p37 was three, slightly higher than that in the PM fraction isolated by the subfractionation procedure.
coverslip surface with polylysine, and sonicated. Recovery of GLUT1 and GRP78 in these membrane fragments was analyzed by SDS–PAGE/immunoblot analysis in comparison to total membranes (Fig. 3). As expected, p46 and GRP78 were detected in total membranes isolated from control cells. In total membranes from glucose-deprived cells, both p37 and p46 were detected along with induced expression of GRP78, as noted in Fig. 1. Plasma membrane fragments from control cells contained p46, but interestingly, no GRP78. Plasma membrane fragments from glucosedeprived cells contained both p46 and p37, but again no GRP78, despite its induced expression. This demonstrates that the plasma membrane fragments were free of intracellular membranes and yet p37 was still observed in fragments from the glucose-deprived cells. This further indicates that the comigration of GRP78 in the subfractionation procedure represents contamination with ER membranes. The ratio of p46:p37 in total membranes was similar to that in PM fragments (2.2 vs 2.5). The data presented herein shows for the first time that GLUT1, when alternatively glycosylated, is targeted in similar if not identical fashion to the normal GLUT1 glycoform. In fact, the similarity in the ratio of these two forms in the PM fraction relative to the homogenate or total membrane fragments, also suggests that the recycling rates of the p46 and p37 (i.e., the movement from the intracellular storage sites to the PM and back again) are the same. In contrast, others have shown that aglyco GLUT1 (i.e., mutagenized transporter lacking the N-linked glycosylation site) is retained within intracellular vesicles (13). This suggests that the presence of oligosaccharide, even though abnormal, is sufficient to provide the appropriate targeting and recycling information. This appears to be the case as well in the LEC1 CHO cells (48)
Plasma Membrane Fragment Isolation The isolation of plasma membrane fragments has been used for characterizing the assembly of clathrincoated pits which occurs at the inner face of the plasma membrane (44, 45) and the purification of caveolin (46). Recently, the technique has been modified for the image analysis of GLUT4 recruitment to the plasma membrane in response to insulin treatment (47). We therefore undertook this method to generate plasma membrane fragments which are potentially free from contamination of endoplasmic reticulum and Golgi apparatus. Adipocytes grown on glass coverslips were deprived of glucose (or not) for 48 h, cross-linked to the
FIG. 3. Identification of GLUT1 in plasma membrane fragments. Cells were incubated in the presence or absence of 25 mM glucose for 48 h before plasma membrane fragments were isolated as described under Materials and Methods. Proteins in the total membrane samples, along with plasma membrane fragments, were separated by SDS–PAGE and probed for GRP78 and GLUT1 after electrophoretic transfer. These data are representative of three independent experiments.
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which lack N-acetylglucosaminyl transferase I (29) resulting in an abbreviated but normal core structure. The functional status of p37 is unknown at this point because of the technical difficulties in separating p37containing membranes from p46-containing membranes. However, we can speculate that p37 transports glucose with less efficiency than p46 from the following observations. Glucose transport activity increases upon glucose deprivation (19). At 24 h of deprivation, the difference can be as much as 10- to 15-fold higher than controls. However, between 24 to 48 h of deprivation, this activity begins to decay . . . such that at 48 h, almost 40% of the “activated” transport activity is lost. P37 appears at about 18 h becoming an increasingly significant percent of the total pool as time progresses. The accumulation of p37 during extended glucose deprivation (24 to 48 h) is inversely correlated with the increase in transport activity. Secondly, glucosedeprived cells supplemented with fructose, exhibit the same “activated” transport activity as cells deprived of glucose in the absence of fructose (19). However, the appearance and accumulation of p37 is completely prevented. This is likely because fructose supplies the precursors for both N-acetylglucosamine and GDP mannose (both of which are important in core oligosaccharide synthesis). Importantly, the transport activity does not decay after 24 h of deprivation. Together these data argue that p37 has less intrinsic activity than does p46, despite the normal targeting. REFERENCES 1. Bell, G. I., Burant, C. F., Takeda, J., and Gould, G. W. (1993) Structure and function of mammalian facilitative sugar transporters. J. Biol. Chem. 268, 19161–19164. 2. Piper, R. C., Hess, L. J., and James, D. E. (1991) Differential sorting of two glucose transporters expressed in insulin-sensitive cells. Am. J. Physiol. 260, C570 –C580. 3. Fisher, M. D., and Frost, S. C. (1996) Translocation of GLUT1 does not account for elevated glucose transport in glucosedeprived 3T3-L1 adipocytes. J. Biol. Chem. 271, 11806 –11809. 4. Calderhead, D. M., Kitagawa, K., Tanner, L. I., Holman, G. D., and Lienhard, G. E. (1990) Insulin regulation of the two glucose transporters in 3T3-L1 adipocytes. J. Biol. Chem. 265, 13800 – 13808. 5. Satoh, S., Nishimura, H., Clark, A. E., Kozka, I. J., Vannucci, S. J., Simpson, I. A., Quon, M. J., Cushman, S. W., and Holman, G. D. (1993) Use of bismannose photolabel to elucidate insulinregulated GLUT4 subcellular trafficking kinetics in rat adipose cells. J. Biol. Chem. 268, 17820 –17829. 6. Yang, J., and Holman, G. D. (1993) Comparison of GLUT 4 and Glut 1 subcellular trafficking in basal and insulin-stimulated 3T3-L1 cells. J. Biol. Chem. 268, 4600 – 4603. 7. Robinson, L. J., and James, D. E. (1992) Insulin-regulated sorting of glucose transporters in 3T3-L1 adipocytes. Am. J. Physiol. 263, E383–E393. 8. Asano, T., Takata, K., Katagiri, H., Tsukuda, K., Lin, J.-L., Ishihara, H., Inukai, K., Hirano, H., Yazaki, Y., and Oka, Y. (1992) Domains responsible for the differential targeting of glucose transporter isoforms. J. Biol. Chem. 267, 19636 –19641.
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