Golgi-Localized, {gamma}-Ear-Containing, Arf-Binding Protein Adaptors Mediate Insulin-Responsive Trafficking of Glucose Transporter 4 in 3T3-L1 Adipocytes

Lin V. Li and Konstantin V. Kandror

Department of Biochemistry, Boston University School of Medicine, Boston, Massachusetts 02118

Address all correspondence and requests for reprints to: K. V. Kandror, Boston University School of Medicine, Department of Biochemistry, K124D, 715 Albany Street, Boston, Massachusetts 02118. E-mail: kandror{at}biochem.bumc.bu.edu.


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS AND DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Small glucose transporter 4 (Glut4)-containing vesicles represent the major insulin-responsive compartment in fat and skeletal muscle cells. The molecular mechanism of their biogenesis is not yet elucidated. Here, we studied the role of the newly discovered family of monomeric adaptor proteins, GGA (Golgi-localized, {gamma}-ear-containing, Arf-binding proteins), in the formation of small Glut4 vesicles and acquisition of insulin responsiveness in 3T3-L1 adipocytes. In these cells, all three GGA isoforms are expressed throughout the differentiation process. In particular, GGA2 is primarily present in trans-Golgi network and endosomes where it demonstrates a significant colocalization with the recycling pool of Glut4. Using the techniques of immunoadsorption as well as glutathione-S-transferase pull-down assay we found that Glut4 vesicles (but not Glut4 per se) interact with GGA via the Vps-27, Hrs, and STAM (VHS) domain. Moreover, a dominant negative GGA mutant inhibits formation of Glut4 vesicles in vitro. To study a possible role of GGA in Glut4 traffic in the living cell, we stably expressed a dominant negative GGA mutant in 3T3-L1 adipocytes. Formation of small insulin-responsive Glut4-containing vesicles and insulin-stimulated glucose uptake in these cells were markedly impaired. Thus, GGA adaptors participate in the formation of the insulin-responsive vesicular compartment from the intracellular donor membranes both in vivo and in vitro.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS AND DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
INSULIN STIMULATES GLUCOSE transport into fat and skeletal muscle cells by translocating glucose transporter isoform Glut4 (glucose transporter 4) to the plasma membrane. In basal cells, Glut4 is accumulated in small 60–80 sedimentation coefficient membrane vesicles that are likely to derive from the endosomal or trans-Golgi donor membranes with a much higher sedimentation coefficient. Several lines of evidence strongly suggest that Glut4-containing small vesicles do not represent an artifact of homogenization and exist in the living cell as an individual insulin-responsive compartment. For example, they are detected by immunoelectron microscopy on ultrathin sections (1, 2, 3, 4) as well as by biochemical methods in extracts of nonhomogenized adipocytes (5). Small Glut4 vesicles are formed in differentiating adipocytes simultaneously with the acquisition of insulin responsiveness (Ref. 6 ; and Shi, J., and K. V. Kandror, in preparation). In addition, small Glut4 vesicles can be formed in vitro from heavy intracellular donor membranes in an ATP-, cytosol-, time-, and temperature-dependent fashion (7).

The question then arises as to what is the mechanism of formation of small insulin-responsive Glut4 vesicles? According to a current model, formation of vesicles is driven by protein coats that are recruited to specific sites on donor membranes by adaptor proteins. In turn, the latter associate with the donor membranes through multiple interactions with the cytoplasmic tails of cargo proteins, Arf- and phosphatidylinositol phosphates (8). Published evidence suggests that adaptor protein (AP)1 or, less likely, AP3 may participate in the formation of Glut4 vesicles on intracellular donor membranes (9, 10, 11, 12). However, as has been recently shown, AP1 may perform its biological functions in cooperation with another family of adaptors called GGAs for Golgi-localized, {gamma}-ear-containing, Arf-binding proteins (13). Therefore, we decided to explore the role of GGA in biogenesis of Glut4 vesicles.

Previously, we have shown that GGAs are involved in targeting of de novo-synthesized Glut4 from the biosynthetic pathway directly to the insulin-responsive compartment bypassing the plasma membrane (14). The question that we are asking in this paper is whether or not GGAs participate in insulin-sensitive recycling of the presynthesized pool of Glut4.

We found that the intracellular localization of GGA2 significantly overlaps with that of Glut4 in the perinuclear area of the cell that may represent either recycling endosomes or trans-Golgi reticulum or both. We also found that Glut4 endocytosed from the plasma membrane is delivered to the perinuclear GGA-containing compartment. In addition, GGA adaptors interact with immunopurified Glut4 vesicles, and dominant negative GGA inhibits formation of these vesicles in vitro. Finally, stable expression of low levels of dominant negative GGA in 3T3-L1 adipocytes inhibits formation of the insulin-responsive Glut4 vesicles in living cells and locks Glut4 in a donor membrane compartment(s). Thus, our results suggest that GGA adaptors not only participate in the delivery of de novo synthesized Glut4 to the insulin-responsive compartment, but also in the regeneration of this compartment after insulin removal. In particular, our data suggest that GGA adaptors are directly responsible for the formation of insulin-responsive Glut4-containing vesicles.


    RESULTS AND DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS AND DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Figure 1AGo shows, by Northern blotting, that GGA1 and GGA2 mRNA are expressed in 3T3-L1 cells throughout the differentiation process. The amount of GGA3 mRNA in differentiating 3T3-L1 cells is below the detection limit of Northern blotting but could be identified by PCR (Fig. 1Go, B and C). In particular, Fig. 1CGo demonstrates that levels of GGA3 mRNA do not change in differentiating cells to a significant degree.



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Fig. 1. Expression of GGA Isoforms in Differentiating 3T3-L1 Adipocytes

A, Total RNA was isolated from 3T3-L1 cells each day of differentiation and analyzed by Northern blotting (20 µg per lane) with the 32P-labeled murine gga1 and gga2 probes. B, Total RNA was isolated from differentiated 3T3-L1 adipocytes, and levels of GGA1 and GGA3 mRNA were determined by RT-PCR. C, Total RNA was isolated from undifferentiated (Fb) and differentiated (Ad) 3T3-L1 cells, and levels of GGA3 mRNA were determined by RT-PCR. mGGA, Murine GGA.

 
To determine whether or not GGA adaptors are involved in insulin-regulated Glut4 traffic, we have electroporated GGA2-enhanced green fluorescent protein (EGFP) cDNA into 3T3-L1 adipocytes that stably express myc7-Glut4. We chose GGA2 for these experiments because this isoform, unlike GGA1 and GGA3, does not have an autoinhibitory DXXLL sequence that can interfere with binding to endogenous ligands (15). Double immunofluorescence staining demonstrates a significant colocalization between GGA2 and Glut4 in the perinuclear area of the cell (Fig. 2Go).



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Fig. 2. Colocalization of GGA2 and Glut4 in Basal 3T3-L1 Adipocytes

3T3-L1 adipocytes stably expressing myc7-Glut4 were electroporated with the GGA2-EGFP cDNA. Cells were replated and grown overnight. Then, cells were fixed, permeabilized, and stained with anti-myc monoclonal antibody followed by Cy3-conjugated donkey antimouse IgG.

 
Recently, we showed that GGA adaptors are responsible for targeting of de novo-synthesized Glut4 to the insulin-responsive compartment (14). Therefore, a fraction of the total intracellular pool of myc7-Glut4 should colocalize with GGA2-EGFP in the biosynthetic pathway. Although it is doubtful that a small amount of novo synthesized Glut4 in the biosynthetic pathway may produce such a strong signal in double immunofluorescence staining (Fig. 2Go), we still wanted to determine directly whether the recycling pool of the transporter is also colocalized with GGA2. For that, we labeled the recycling pool of myc7-Glut4 with the anti-myc antibody at the plasma membrane according to a recently described protocol (16, 17). Briefly, 3T3-L1 adipocytes stably expressing myc7-Glut4 were electroporated with GGA2-EGFP cDNA similar to the previous experiment. Next day, cells were stimulated or not stimulated with insulin, cooled to 0 C to inhibit endocytosis, and incubated with the monoclonal antibody against the myc epitope. Figure 3AGo shows that, in the absence of insulin, the anti-myc antibody does not bind to cells. Insulin administration causes translocation of myc7-Glut4 to the cell surface, thus allowing the anti-myc antibody to bind to the extracellular myc epitope (Fig. 3BGo). Cells were then washed and transferred to 37 C for internalization of the antibody associated with myc7-Glut4. Staining of these cells with rabbit antimouse IgG (Fig. 3CGo) shows that the internalized antibody and, correspondingly, myc7-Glut4 are delivered to the perinuclear GGA2-containing compartment. Analogous experiments in which the monoclonal anti-myc antibody was replaced with nonspecific mouse IgG did not demonstrate any staining and are not shown.



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Fig. 3. Internalized myc7-Glut4 Prebound to Anti-myc Antibody at the Plasma Membrane Is Delivered to the GGA2-Positive Compartment

3T3-L1 adipocytes stably expressing myc7-Glut4 were electroporated with the GGA2-EGFP cDNA and grown overnight. A, Basal cells were cooled to 0 C, incubated on ice with the monoclonal antibody against myc (25 µg/ml) for 1 h, and then washed, fixed, and stained with Cy3-conjugated donkey antimouse IgG. B, Cells were stimulated with 200 nM insulin for 20 min at room temperature, and then transferred on ice and incubated with the monoclonal anti-myc antibody as described above. At the end of the incubation, cells were washed, fixed, and stained with Cy3-conjugated donkey antimouse IgG. C, After incubation with the anti-myc antibody, cells were washed as in panels A and B and transferred to 37 C for 1 h. Cells were then fixed, permeabilized, and stained with Cy3-conjugated donkey antimouse IgG.

 
To get insight into the nature of the GGA-positive perinuclear compartment in adipocytes, we created a 3T3-L1 cell line stably expressing GGA2-EGFP using the retroviral expression vector pLNCX2. The intracellular distribution of this protein showed a significant overlap with endocytosed transferrin and syntaxin 6 (18, 19) (Fig. 4Go, A and B). Thus, GGA2 in 3T3-L1 adipocytes, similar to other cell types (20, 21), is localized in the trans-Golgi network and, to some extent, in endosomes. To determine whether or not stably expressed GGA2 colocalizes with Glut4, cells infected with GGA2-EGFP-carrying retrovirus were electroporated with myc7-Glut4 cDNA. Staining with anti-myc antibody demonstrated a significant colocalization of GGA2 with myc7-Glut4 specifically in the perinuclear region of the cell (Fig. 4Go C). Thus, regardless of the experimental design, we detect a major colocalization of Glut4 and GGA2 (compare Figs. 2Go and 4CGo).



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Fig. 4. In 3T3-L1 Adipocytes, GGA2 Is Localized in Endosomes and Trans-Golgi Network

A, 3T3-L1 adipocytes stably expressing GGA2-EGFP were incubated with Texas Red-labeled transferrin for 10 min at 37 C. Cells were then washed, fixed, and examined by double immunofluorescence microscopy. B, 3T3-L1 adipocytes stably expressing GGA2-EGFP were fixed, permeabilized, and stained with monoclonal antibody against syntaxin 6 followed by Cy3-conjugated donkey antimouse IgG. C, 3T3-L1 adipocytes stably expressing GGA2-EGFP were electroporated with the cDNA for myc7-Glut4. Cells were replated, grown overnight, and then fixed, permeabilized, and stained with monoclonal anti-myc antibody followed by Cy3-conjugated donkey antimouse IgG. Tf, Transferrin.

 
It is possible, however, that despite significant colocalization between GGA2 and Glut4, there is no functional connection between these proteins. To explore this issue, we have constructed a dominant negative GGA2 mutant, VHS (Vps-27, Hrs, and STAM)-GAT (GGA and TOM1)-EGFP, in which the clathrin-binding hinge domain and the auxiliary protein-binding {gamma}-adaptin ear domain have been substituted with EGFP. The mutant still has its substrate-binding VHS domain and the Arf-binding GAT domain that are conserved in all three GGA isoforms (21). We suggest, therefore, that our mutant has a dominant negative effect on all three known GGA isoforms. This construct was stably expressed in 3T3-L1 cells with the help of the retroviral expression vector pLNCX2. Visual analysis of the individual stable clones showed that the level of VHS-GAT-EGFP expression was, at best, moderate, suggesting a toxic effect of the dominant negative mutant. To estimate the level of VHS-GAT-EGFP expression, we have compared Western blot EGFP signals from the total cell lysate and purified EGFP (Fig. 5AGo). Considering that one P100 Petri dish with differentiated 3T3-L1 adipocytes contains 3 x 106 cells and yields approximately 3 mg of the total protein, we estimate that transfected cells have about 100,000 copies of VHS-GAT-EGFP per cell. For comparison, a differentiated 3T3-L1 cell has approximately 300,000 Glut4 molecules (22). It was reported that prolonged culture of GGA-silenced cells causes cell death (23). However, such moderate quantities of VHS-GAT-EGFP are well tolerated by 3T3-L1 cells that do not show any changes in the rate of growth and differentiation in comparison with empty vector-transfected cells. Figure 5BGo demonstrates that ectopically expressed VHS-GAT-EGFP has a punctate perinuclear distribution similar to the endogenous wild-type GGA proteins (24, 25, 26). Expression of VHS-GAT-EGFP leads to a small increase in the total Glut4 level (Fig. 5CGo) that may or may not represent a compensatory mechanism in the cell.



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Fig. 5. Expression of the Dominant Negative Construct VHS-GAT-EGFP in 3T3-L1 Cells

A, Total lysate (300 µg) from VHS-GAT-EGFP expressing 3T3-L1 adipocytes and purified recombinant EGFP were analyzed by Western blotting on the same membrane using the antibody against EGFP. B, 3T3-L1 adipocytes stably expressing VHS-GAT-EGFP were examined by fluorescence microscopy. C, Expression levels of Glut4 and Glut1 in empty vector infected (EV) and VHS-GAT-EGFP expressing (DN) 3T3-L1 adipocytes were analyzed by Western blotting (100 µl of total cell lysate per lane).

 
VHS-GAT-EGFP-transfected cells demonstrate a marked decrease in maximal insulin-stimulated glucose uptake (Fig. 6AGo). This suggests that the Glut4 pathway may be more sensitive to VHS-GAT-EGFP than other intracellular trafficking events because the dominant negative mutant inhibits insulin-stimulated glucose uptake before manifestation of its toxic effect.



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Fig. 6. Dominant Negative GGA-Expressing Adipocytes Have Lower Insulin-Stimulated Glucose Uptake than Control Cells

A, Empty vector-infected (white bars) and VHS-GAT-EGFP-expressing (gray bars) 3T3-L1 adipocytes were serum starved for 2 h, and insulin (100 nM) was added for 15 min at 37 C. Measurements of [3H]2-deoxyglucose uptake were performed in duplicate as described in Materials and Methods. B, Empty-vector infected (dotted line) and VHS-GAT-EGFP-expressing (solid line) 3T3-L1 adipocytes were stimulated with 100 nM insulin for 15 min. Insulin was removed by washing five times with cold DMEM, and [3H]2-deoxyglucose uptake was measured at indicated time points. C, Cells were treated as described in panel B. After insulin removal, cells were re-stimulated with 100 nM insulin at indicated time points, and the uptake of [3H]2-deoxyglucose was determined. In panels B and C, arbitrary units were defined as glucose uptake (in picomoles of 2-DOG/min·mg protein) normalized by the maximal glucose uptake measured in starved cells after stimulation with 100 nM insulin for 15 min. All panels show representative results of three independent experiments. DOG, Deoxyglucose; a.u., arbitrary units.

 
Insulin-stimulated glucose uptake may be affected by the rate of Glut4 internalization as well. However, this should not be the case in VHS-GAT-EGFP-transfected cells, because, upon insulin removal, glucose transport is reversed at a normal rate and reaches the basal level in 2 h (Fig. 6BGo). To study the effect of VHS-GAT-EGFP on the reformation of the insulin-responsive compartment upon insulin withdrawal, cells were stimulated with 100 nM insulin for 15 min, and insulin was then removed by five washes with DMEM. After a 30-, 60-, and 120-min recovery, insulin-stimulated glucose uptake was measured again and expressed as a percentage of the insulin-stimulated rate (Fig. 6CGo). In control empty vector-transfected cells, full insulin response was reestablished after a 120-min recovery. On the contrary, VHS-GAT-EGFP-transfected cells cannot reform the insulin-sensitive compartment within this time period. Because the rate of Glut4 internalization in both cell lines is the same, we think that VHS-GAT-EGFP inhibits formation of the insulin-responsive compartment in 3T3-L1 cells.

From the biochemical standpoint, the insulin-responsive compartment represents small 60–80 sedimentation coefficient membrane vesicles, or IRVs (insulin-responsive vesicles), that can be readily separated from heavy donor membranes by centrifugation at 16,000 x g (5, 7). We found that, in VHS-GAT-EGFP-expressing cells, significantly more Glut4 is localized in rapidly sedimenting donor membranes and less Glut4 is present in the 16,000 x g supernatant where small insulin-responsive vesicles are recovered (Fig. 7Go, A and B). At the same time, the sedimentation coefficients of small Glut4 vesicles in VHS-GAT-EGFP-transfected and control cells are identical (results not shown).



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Fig. 7. Dominant Negative GGA-Expressing Adipocytes Have Less Glut4 in the Insulin-Sensitive Vesicular Fraction than Control Cells

Empty vector-infected (EV) and VHS-GAT-EGFP-expressing (DN) 3T3-L1 adipocytes were homogenized and subjected to the 16,000 x g centrifugation. The pellet and supernatant (100 µg each) were analyzed by Western blotting. A, Results of a representative experiment. B, Normalized mean values ± SE of three independent experiments. P, Pellet; S or sup, supernatant.

 
These results suggest that GGA adaptors are involved in the formation of the IRVs. However, Glut4 does not have the DXXLL sequence recognized by the VHS domain of GGA adaptors. Correspondingly, we were unable to detect a direct interaction between Glut4 and GGA2 (results not shown). It is possible, however, that GGAs participate in the formation of Glut4 vesicles via some other vesicular protein. One possibility is that this putative protein is sortilin. Sortilin represents a major component of small Glut4 vesicles (27, 28) and has a canonical DXXLL GGA-recognition sequence in its cytoplasmic tail (29).

To determine whether or not GGA can interact with Glut4 vesicles, we immunoadsorbed them from VHS-GAT-EGFP-transfected cells with the help of the monoclonal 1F8 antibody raised against the C terminus of Glut4. We found that VHS-GAT-EGFP is coimmunoadsorbed with Glut4 vesicles (Fig. 8AGo), suggesting that GGA adaptors not only colocalize with Glut4 vesicles (Figs. 2Go and 4CGo), but also physically interact with this compartment. To confirm this result, we performed the glutathione-S-transferase (GST)-pull-down assay using the recombinant VHS domain. In agreement with Fig. 8AGo, Glut4 vesicles from 3T3-L1 (Fig. 8BGo) and primary (Fig. 8CGo) adipocytes efficiently interact with this domain. Importantly, the VHS domain, which lacks 33 amino acids and is thus unable to bind the DXXLL sequence (20, 30), does not interact with Glut4 vesicles (Fig. 8BGo). This result is consistent with the idea that sortilin mediates binding of GGA adaptors to Glut4 vesicles.



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Fig. 8. Glut4-Containing Vesicles Interact with GGA Adaptors in Vitro

A, Protein A-purified 1F8 antibody and nonspecific mouse IgG were prebound to sheep antimouse Dynabeads and incubated with 16,000 x g supernatant (100 µg) from empty vector-infected (EV) and VHS-GAT-EGFP-expressing (DN) 3T3-L1 adipocytes. Material bound to the beads was subjected to Western blot analysis. B, GST alone, GST fused with the wild-type VHS domain of GGA2 (GST-VHS), and GST fused with the VHS domain lacking amino acids 68–101 (GST-VHS{Delta}68–101) were prebound to GSH-Sepharose beads. 3T3-L1 adipocytes were homogenized and separated into pellet and supernatant by 16,000 x g centrifugation. The latter fraction (100 µg) was incubated with the beads as described in Materials and Methods, and material bound to the beads was subjected to Western blot analysis. C, The pull-down experiment with GST and GST-VHS was performed as described above using material prepared from primary rat adipocytes. WB, Western blot; IP, immunoprecipitation.

 
Our data show that the dominant negative GGA mutant inhibits formation of the IRVs in stably transfected cells (Fig. 7Go). To reconstitute this result in vitro, we used a cell-free vesicle budding assay recently developed in our laboratory (7). We found that cytosol from empty vector-transfected cells efficiently supports formation of small Glut4 vesicles in vitro, whereas cytosol from VHS-GAT-EGFP-expressing cells is not active (Fig. 9AGo).



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Fig. 9. Dominant Negative GGA Inhibits Formation of Small Glut4 Vesicles in Vitro

A, Vesicle reconstitution assay was carried out with donor membranes isolated from wild-type 3T3-L1 adipocytes and cytosol from empty vector-infected (EV) and VHS-GAT-EGFP-expressing (DN) 3T3-L1 adipocytes. The vesicle and the donor membrane fractions were analyzed by Western blotting using 1F8 antibody. B, Vesicle reconstitution assay was carried out in the presence of indicated amounts (in micrograms) of recombinant GST or VHS-GAT-GST.

 
Small Glut4 vesicles represent a mixture of at least two vesicular populations: IRVs and cellugyrin-positive vesicles. The latter are not translocated to the plasma membrane in response to insulin stimulation and mediate Glut4 traffic between different intracellular compartments (5, 31). Although we know that the budding reaction in vitro produces predominantly IRVs (7), we still wanted to determine the effect of the dominant negative GGA mutant on the formation of cellugyrin-positive vesicles. We found that recombinant dominant negative GST-VHS-GAT fusion protein inhibits formation of small Glut4 vesicles in vitro, whereas formation of cellugyrin-containing vesicles is affected to a much lesser degree (Fig. 9BGo). Previously, we have shown that formation of the transferrin receptor- and Glut1-containing vesicles in vitro is resistant to GST-VHS-GAT (14). We believe, therefore, that GGA adaptors are involved primarily in the formation of the IRVs and may not participate in intracellular Glut4 traffic en route to this compartment.

Recently, we have shown that entry of newly synthesized Glut4 into the insulin-responsive storage compartment is GGA dependent (14). This study demonstrates that the role of GGA adaptors is not limited to transport of newly synthesized Glut4 but includes presynthesized recycling Glut4 molecules as well. In fact, data presented in Figs. 3Go and 6CGo directly demonstrate that GGAs are involved in the regeneration of the IRV pool upon insulin withdrawal. Apparently, this pool is replenished not with newly synthesized Glut4 but with presynthesized transporter molecules internalized from the plasma membrane. In addition, de novo synthesized Glut4 cannot account for the significant inhibitory effect of the cytosol from VHS-GAT-EGFP transfected cells on the formation of Glut4 vesicles in vitro (Fig. 9AGo). We suggest, therefore, that the Glut4 biosynthetic pathway may overlap with the Glut4 recycling pathway in either an endosomal compartment or in the trans-Golgi network (Fig. 10Go). According to this model, newly synthesized and presynthesized Glut4 molecules may enter the same GGA-formed IRVs.



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Fig. 10. GGA Adaptors Participate in the IRV Recruitment of Both Novo-Synthesized (14 ) and Presynthesized (this Paper) Glut4 Molecules

The black arrow indicates the biosynthetic pathway; white arrows indicate the Glut4 recycling pathway. ER, Endoplasmic reticulum; TGN, trans-Golgi network.

 

    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS AND DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Materials
We used monoclonal anti-Glut4 antibody 1F8 (32), monoclonal antibody against syntaxin 6 (BD Biosciences, Palo Alto, CA), monoclonal antibody against EGFP sc-9996 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA), monoclonal anti-myc antibody 9B11 (Cell Signaling Technology, Beverly, MA), and the polyclonal antibody against GST G7781 (Sigma Chemical Co., St. Louis, MO). Cy3-conjugated donkey antimouse IgG was from Jackson ImmunoResearch Laboratories, Inc (West Grove, PA). Dexamethasone, 3-isobutyl-1-methylxanthine, insulin, benzamidine, potassium aspartate, potassium glutamate, potassium gluconate, 3[N-morpholino]propane sulfonic acid, sodium carbonate, magnesium sulfate, glutathione (reduced form), ATP, creatine phosphate, creatine phosphokinase, brefeldin A (BFA), Wortmannin, guanosine 5'-({gamma}-thio)triphosphate, mouse IgG, and rabbit IgG were purchased all from Sigma. Aprotinin, leupeptin, pepstatin A, and phenylmethylsulfonyl fluoride were obtained from American Bioanalytical (Natick, MA). Radiolabeled 2-deoxyglucose was obtained from PerkinElmer Life Sciences (Norwalk, CT). G418, calf bovine serum, fetal bovine serum, and DMEM were purchased from Invitrogen (San Diego, CA). Recombinant EGFP was from BD Biosciences. cDNAs for human GGA3 and GGA3{Delta}68–101 were kindly provided by Dr. Bonifacino (National Institutes of Health).

Plasmids
Murine gga2 cDNA was obtained by RT-PCR of 3T3-L1 adipocyte mRNA with the following primers: 5'-GGGTACCTTTTGGGCAGGAGTGACTG-3' and 5'-GTATGCGGCCGCACAACAAGTCCCGACCAGAC-3' designed according to gga2 cDNA sequence (GenBank accession no. XM_133801) and cloned into the pGEM-T vector (Promega Corp., Madison, WI). For expression in bacteria, gga2 VHS domain (residues 1–188) and VHS-GAT fragment (residues 1–325) were subcloned into the pGEM-4T-3 (Amersham Biosciences, Arlington Heights, IL) vector using EcoRI-XhoI and EcoRI-SalI restriction sites correspondingly. For expression in mammalian cells, gga2 and its VHS-GAT fragment were subcloned into the pEGFP-N3 vector using BglII-SalI and BglII-KpnI restriction sites. The GFP-tagged GGA2 and the VHS-GAT fragment were then subcloned into BglII-NotI sites of the retroviral vector pLNCX2. VHS domains of human GGA3 and GGA3{Delta}68–101were subcloned into the pGEM-4T-3 vector using EcoRI-XhoI restriction sites.

Cell Culture and Retroviral Transfection
Murine 3T3-L1 preadipocytes were cultured, differentiated, and maintained as described previously (33). Briefly, cells were grown in DMEM supplemented with 10% calf bovine serum until confluence. The cells were transferred 2 d later to differentiation medium (DMEM containing 10% fetal bovine serum, 0.5 mM 3-isobutyl-1-methylxanthine, 1 µM dexamethasone, and 1.7 µM insulin). After 48 h, the differentiation medium was replaced with maintenance medium (DMEM supplemented with 10% fetal bovine serum). The maintenance medium was changed every 48 h. The cells were used at d 8 of differentiation. For stable expression of GGA2-EGFP and VHS-GAT-EGFP constructs in 3T3-L1 cells, pLNCXII-VHS-GAT-EGFP or pLNCXII vectors (20 µg each) were first added to PT67 packaging cells at 70–80% confluency in a P100 Petri dish for 48 h. The medium was then replaced with 5.5 ml DMEM with 10% fetal bovine serum. After another 48 h, the virus-containing medium was collected, filtered through a 0.45-µm filter and added, together with 4 µg/ml polybrene, to a P100 Petri dish with 3T3-L1 preadipocytes at 20–30% confluency for 8 h. After a 48-h recovery period, infected cells were selected in DMEM containing 10% calf bovine serum and 400 µg/ml G418. Clones of G418-resistant cells were combined and used for the following experiments. Stable transfection of 3T3-L1 cells with pBabe-myc7-Glut4 was described previously (34).

Electroporation
3T3-L1 adipocytes grown in a 10-cm Petri dish were trypsinized, washed twice with PBS, and re-suspended in 0.5 ml of PBS. Plasmid DNA (100 µg) and adipocyte cell suspension were mixed in a gene pulser cuvette with a 0.4-cm electrode gap. Electroporation was performed with the help of a Bio-Rad Gene Pulser (Bio-Rad Laboratories, Inc., Hercules, CA) with 960-µF capacitance at 0.16 kV for 16–18 msec. After gene transfer, cells were transferred to 4 ml of DMEM with 10% calf serum for 10 min for recovery at room temperature and then replated on cover slips.

Glucose Uptake
[3H]2-deoxyglucose uptake into differentiated 3T3-L1 cells was measured in six-well plates. Briefly, cells were preincubated in serum-free DMEM for 2 h, and the medium was changed to 900 µl/well Krebs-Ringer HEPES (121 mM NaCl; 4.9 mM KCl; 1.2 mM MgSO4; 0.33 mM CaCl2; 12 mM HEPES, pH 7.4) with or without 100 nM insulin for 15 min. [3H]2-deoxyglucose (final concentration, 0.12 mM, 4.4 mCi/mmol) was added for another 4 min. The reaction was terminated by the addition of 1 ml of cold Krebs-Ringer HEPES with 25 mM D-glucose. Cells were washed, lysed with 0.1% sodium dodecyl sulfate, and radioactivity was counted in an LKB scintillation counter (LKB, Rockville, MD). Carrier-specific uptake was obtained by correction for nonspecific diffusion of [3H]2-deoxyglucose into the cells in the presence of 10 µM cytochalasin B.

GST Fusion Protein Purification
Expression of GST and GST-VHS fusion protein was induced in BL21 cells (Stratagene, La Jolla, CA) by 1 mM isopropyl-b-D-thiogalactopyranoside (Sigma) for 3 h at 37 C. Expression of GST-VHS-GAT was induced by 0.1 mM isopropyl-b-D-thiogalactopyranoside for 4 h at 20 C to avoid formation of inclusion bodies. Cells were lysed with lysozyme (Sigma) and treated with DNAse and RNAse (Invitrogen). Insoluble debris was removed by centrifugation at 3000 x g for 30 min at 4 C, and GST-containing proteins were purified from the supernatant by affinity chromatography using glutathione-Sepharose 4B (Amersham Biosciences, Piscataway, NJ) according to manufacturer’s instructions.

GST Pull-Down Assay
3T3-L1 adipocytes were homogenized in PBS with the Protease Inhibitor cocktail (Roche Clinical Laboratories, Indianapolis, IN) and centrifuged at 16,000 x g for 20 min. The supernatant (100 µg) was incubated with 10 µg of GST or GST-VHS and 50 µl (50% slurry in PBS) of glutathione-conjugated Sepharose 4B beads (Amersham Biosciences) for 4 h at 4 C. Beads were collected by centrifugation at 500 x g for 5 min, washed five times with PBS, eluted with an equal volume of 2x sample buffer, and separated by SDS-PAGE.

Immunoadsorption Experiments
Protein A-purified 1F8 antibody and nonspecific mouse IgG (2 mg each) were bound to 30 µl Dynabeads M-280 sheep antimouse IgG (Dynal Biotech, Great Neck, NY) according to manufacturer’s instructions. Before usage, the beads were preincubated with 0.1% BSA in PBS for at least 1 h and rinsed with PBS. The 16,000 x g supernatant (100 µg) from 3T3-L1 adipocytes was incubated with specific and nonspecific antibody-coupled beads separately overnight at 4 C. The beads were washed five times with PBS, and the adsorbed material was eluted with equal volume of 2x sample buffer and separated by SDS-PAGE.

In Vitro Vesicle Reconstitution Assay
3T3-L1 cells were incubated in serum-free media for 2 h, homogenized in a ball-bearing cell cracker (European Molecular Biology Laboratory) and centrifuged at 16,000 x g for 20 min. The supernatant was collected and centrifuged again at 200,000 x g for 60 min. The budding reaction mixture consisted of the pellet of the 16,000 x g centrifugation (donor membranes, 250 µg), the supernatant of the 200,000 x g centrifugation (cytosol, 500 µg) and the ATP regeneration system (1 mM ATP, 8 mM creatine phosphate, 1.5 U/ml creatine phosphokinase) as previously described (7). The samples were then transferred to 37 C for 20 min and centrifuged at 16,000 x g to pellet donor membranes and then at 200,000 x g for 60 min. The pellet of the 200,000 x g centrifugation that contained de novo formed vesicles was subjected to Western blot analysis.

Gel Electrophoresis and Immunoblotting
Proteins were separated by SDS-PAGE according to Laemmli (35) and transferred to a polyvinylidine difluoride membrane in 25 mM Tris, 192 mM glycine. After transfer, the membrane was blocked with 10% nonfat dry milk in PBS for 1 h at 25 C and probed with specific antibodies overnight. The membranes were washed three times with PBS-0.05% Tween and incubated with horseradish peroxidase-labeled secondary antibody for 1 h at room temperature. After three more washes the membranes were incubated in enhanced chemiluminescence reagent (New England Nuclear, Boston, MA) for 1 min and then exposed to a Kodak 440 image station Eastman Kodak (Rochester, NY). Data analysis was performed with Kodak 1D image analysis software.

Northern Blot Analysis and RT-PCR
3T3-L1 cells were differentiated as described above, and total RNA was isolated each day of differentiation from one P100 Petri dish using TRIzolLS (Invitrogen) as recommended by the manufacturer. Total RNA (20 µg) was electrophoresed in a 1% agarose/2% formaldehyde gel followed by transfer and UV cross-linking to a Hybond-n + membrane (Amersham Biosciences). Murine gga1 probe (501 bp) was obtained by RT-PCR using primers 5'-CTGAACAAGGAGCTGAACTG-3'and 5'-AGGGTGTGAGCTCTTCAGCA-3'. Murine gga2 probe (532bp) was obtained by PCR from the pGEM-T-GGA2 vector using primers 5'-ATGGCAGCGACGGCAGTG-3' and 5'-GGGGGCAAGATTTTATCCAT-3'. Murine gga3 probe (513 bp) was obtained by RT-PCR using primers 5'-CAACAAAGAGCTTGAAGGGC-3' and 5'-CGAGCCTCATCTTCCTTCAAC-3'. Hybridization was performed in ExpressHyp solution (CLONTECH Laboratories, Inc., Palo Alto, CA) as recommended by the manufacturer. RT-PCR was performed with the help of the SuperScript one-step RT-PCR kit from Invitrogen using the same primers.

Immunocytochemistry
3T3-L1 adipocytes were lifted and grown on coverslips coated with poly-L-lysine overnight. Cells were fixed with 4% paraformaldehyde in PBS for 15 min and permeabilized (where indicated) using PBS with 0.2% Triton. Cells were washed with PBS and incubated in blocking solution (PBS with 5% BSA and 5% donkey serum) for 1 h at room temperature. Cells were labeled with primary and then with secondary antibodies and mounted on slides using the SlowFade-Light Antifade kit (Molecular Probes, Inc., Eugene, OR). Cells were examined with the help of an Axiovert 200M fluorescence microscope and an Axiovision 3.0 program (Carl Zeiss, Inc., Thornwood, NY) or with the help of a confocal laser scanning microscope LSM510 (Carl Zeiss, Inc.).

Transferrin Uptake
Cells were washed twice with DMEM and incubated in DMEM supplemented with Texas Red-labeled human transferrin (5 µg/ml) at 37 C. Cells were then washed with cold DMEM, fixed, and examined as described in the previous section.

Antibody Internalization Assay
Electroporated 3T3-L1 adipocytes were grown on cover slips coated with collagen (Roche) overnight. Cells were stimulated with 200 nM insulin for 20 min, and then transferred on ice and washed with ice-cold DMEM containing 0.2% BSA. Cold cells were incubated with either monoclonal anti-myc antibody or nonspecific mouse IgG (both 25 µg/ml in DMEM with 0.2% BSA) for 1 h on ice. At the end of the incubation, cells were washed extensively and either stained without permeabilization using Cy3-conjugated donkey antimouse antibody or transferred to 37 C for another hour followed by fixation, permeabilization and staining with the same antibody.

Statistical Analysis
Student’s unpaired two-tailed t test was used to evaluate statistical significance of the results.


    ACKNOWLEDGMENTS
 
We thank Jun Shi for providing the 3T3-L1 cell line stably expressing myc-Glut4, Dr. Bonifacino for hGGA3 and hGGA3{Delta}68–101 constructs, Dr. Roland Govers for valuable technical advice, and Dr. Galini Thoidis for help with the manuscript.


    FOOTNOTES
 
This work was supported by Research Grants DK52057 and DK56736 from the National Institutes of Health and by a research grant from the American Diabetes Association (to K.V.K.)

First Published Online March 17, 2005

Abbreviations: AP1, Adaptor protein 1; EGFP, enhanced green fluorescent protein; GAT, GGA and TOM1; GGA, Golgi-localized, {gamma}-Ear-containing, Arf-binding protein; Glut4, glucose transporter 4; GST, glutathione-S-transferase; IRV, insulin-responsive vesicle; VHS, Vps-27, Hrs, and STAM.

Received for publication January 12, 2005. Accepted for publication March 9, 2005.


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