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Vol. 17, Issue 5, 2303-2311, May 2006
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* Life Sciences Institute, Department of Internal Medicine, University of Michigan, Ann Arbor, MI 48109;
Department of Molecular and Integrative Physiology, University of Michigan, Ann Arbor, MI 48109
Submitted January 13, 2006;
Revised February 24, 2006;
Accepted February 28, 2006
Monitoring Editor: Adam Linstedt
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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; Bryant et al., 2002). In unstimulated cells, Glut4 undergoes endocytosis into endosomes and subsequently sorts into specialized storage vesicles that traffic to the plasma membrane after activation of the insulin receptor. The vesicles then dock and fuse at specific sites at the membrane, resulting in extracellular exposure of the transporter. The precise signals from the insulin receptor that control these events involve the tyrosine phosphorylation of a number of intracellular substrates. These phosphorylations lead to activation of the PI3-kinase pathway (Saltiel and Kahn, 2001) and also to activation of the G protein TC10 (Chiang et al., 2001; Watson et al., 2001; Maffucci et al., 2003), which in turn binds to numerous effectors, including the exocyst protein Exo70 (Inoue et al., 2003). Exo70 exists in a multiprotein complex with the proteins Sec6 and Sec8. A dominant-negative mutant of Exo70 blocked insulin-stimulated glucose uptake, but was without effect on translocation of Glut4 to the plasma membrane. However, this Exo70 mutant prevented the appearance of Glut4 at the cell surface, leading us to propose that the exocyst complex may play a critical role in tethering Glut4 vesicles to the plasma membrane for subsequent docking and fusion (Inoue et al., 2003).
Lipid rafts are specialized compartments of the plasma membrane enriched in cholesterol and glycosphingolipids. Numerous signaling and cytoskeletal proteins are found in these subdomains, suggesting that they may act as organizing centers for signal transduction, particularly for insulin (Anderson, 1998; Schlegel et al., 1998; Watson and Pessin, 2001; Bickel, 2002; Saltiel and Pessin, 2002, 2003). Both the insulin receptor and TC10 reside in lipid rafts (Yamamoto et al., 1998; Gustavsson et al., 1999; Nystrom et al., 1999; Watson et al., 2001; Kimura et al., 2002; Vainio et al., 2002), and the mistargeting of TC10 to a nonlipid raft domain prevents its activation by insulin and blocks insulin action (Watson et al., 2001; Kanzaki and Pessin, 2002; Chunqiu Hou and Pessin, 2003; Watson et al., 2003).
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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antibody were obtained from Santa Cruz Biotechnology (Santa Cruz, CA), and anti-GFP polyclonal antibody was obtained from Abcam (Cambridge, MA). Anti-total Akt and phospho-Akt antibodies were from Cell Signaling (Beverly, MA). Anti-Sec6, Sec8, and SAP97 monoclonal antibodies were from Stressgen (San Diego, CA). Anti-caveolin polyclonal antibody and anti-caveolin2 mAb were from BD Transduction Laboratory (San Jose, CA). Anti-Glut4 polyclonal antibody was from Alpha Diagnostic International (San Antonio, TX). Anti-Exo70 mAb kindly provided from Dr. Shu-Chan Hsu (Rutgers University; Vega and Hsu, 2001).
Cell Culture and Electroporation of 3T3L1 Adipocytes
The 3T3L1 fibroblasts were grown in DMEM with 10% fetal bovine serum (FBS) and differentiated into adipocytes as described (Baumann et al., 2000). The 3T3L1 adipocytes were transfected with stealth RNA interference (RNAi) duplexes (Invitrogen, Carlsbad, CA) by electroporation. In brief, adipocytes at day 2 of differentiation were detached from culture dishes with 0.25% trypsin, washed twice, and resuspended in phosphate-buffered saline (PBS). Approximately 5 x 106 cells (half of the cells from one p150 dish) were mixed with RNAi duplexes, which were delivered to the cells by electroporation with a Bio-Rad gene pulser II system (Richmond, CA; 0.16 kV and 960 µF). After electroporation, cells were mixed with medium for 10 min before replating.
Immunoprecipitation and Immunoblotting
Cells were lysed with HNTG buffer using 1% Triton X-100. Lysates were incubated with the indicated antibodies for 2 h at 4°C. The immune-complexes were precipitated with protein A or G agarose beads (Amersham Pharmacia, Piscataway, NJ) and washed with lysis buffer. The samples were resolved in 4-20% gradient SDS-PAGE and analyzed by immunoblotting. All immunoblots were developed by enhanced chemiluminesence (Amersham Pharmacia).
Immunofluorescence Microscopy
For immunofluorescence studies, cells were processed as described (Kimura et al., 2001, 2002; Liu et al., 2005). To make plasma membrane sheets, cells were treated for 5 min with the Triton extraction buffer (50 mM MES, pH 6.0, 150 mM NaCl, 0.25% Triton X-100, 1 mM CaCl2, 0.5 mM MgCl2) before fixation. To detect myc-Exo70, cells were stained with anti-myc mAb or polyclonal antibody (Santa Cruz Biotechnology). To detect HA-TC10, cells were stained with anti-HA mAb (Santa Cruz Biotechnology). After incubation with primary antibodies, cells were incubated with Alexa488 or Alexa594 goat anti-mouse or anti-rabbit immunoglobulin (IgG), obtained from Molecular Probes (Invitrogen, Eugene, OR). Images were captured by using an Olympus FV300 confocal laser scanning microscope (Melville, NY).
Expression Constructs
To generate a mammalian expression vector, full-length Sec8 was subcloned into the pKH3 vector. YFP-mycSAP97 construct was a gift from Dr. Benjamin Margolis (University of Michigan). The His-tagged proteins Sec8 and its 
4aa deletion mutant were cloned into a pET15b vector.
2-Deoxyglucose Uptake Assay
The RNAi-transfected cells were reseeded on 12-well plates and cultured for 4 d. After incubation with DMEM containing 0.5% FBS for 3 h, cell were washed with Krebs-Ringer buffer (130 mM NaCl, 5 mM KCl, 1.3 mM CaCl2, 1.3 mM MgSO4, 25 mM HEPES, pH 7.4) and incubated with or without insulin for 30 min. Glucose uptake was initiated by addition of [14C]2-deoxy-D-glucose to a final assay concentration of 100 µM for 5 min and terminated by three washes with ice-cold PBS, the cells were solubilized with 0.1% SDS, and [14C] was determined by scintillation counting.
Sucrose Gradient Centrifugation
3T3L1 adipocytes were homogenized in HES buffer (20 mM HEPES, pH 7.5, 250 mM sucrose, 1 mM EDTA) with a Dounce homogenizer. After homogenization, cells were centrifuged at 800 x g and the supernatant was centrifuged at 15,000 x g to produce a total plasma membrane fraction. Total plasma membrane fraction was solubilized in 0.3 ml of 0.2% Triton X-100 in MBS, and made up to 0.4 ml with MBS. These samples were mixed with 0.4 ml of 80% sucrose (wt/vol), and overlaid with 3.5 ml of 30%, 1 ml of 5% sucrose. The gradient was centrifuged at 150,000 x g for 19 h, and 440-µl fractions were collected from the top of each gradient.
PDZ Domain Array
His-tagged Sec8 wild-type and Sec84aa mutant protein were prepared as previously described (Taylor et al., 2000). TranSignal PDZ Domain Arrays (Panomics, Redwood City, CA) were used as instructed.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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; Kee et al., 1997; Lipschutz and Mostov, 2002; Yeaman, 2003; Guo and Novick, 2004; Hsu et al., 2004; Tsuboi et al., 2005). We sought to evaluate whether exocyst proteins play a crucial role in insulin-stimulated Glut4 vesicle trafficking and glucose transport. We knocked down components of the exocyst complex by electroporation of 3T3L1 adipocytes with RNAi oligomers and assayed insulin-stimulated 2-deoxyglucose uptake (Figure 1, A and B). In 3T3L1 adipocytes transfected with a scrambled oligomer, insulin produced an eightfold increase in glucose uptake. Knockdown of total Exo70 protein by 50-70% inhibited insulin-stimulated glucose transport by 
50% compared with the control scrambled RNAi oligo. This inhibitory effect of knocking down Exo70 was seen at all concentrations of insulin. Similar results were observed using two other RNAi oligos targeting two distinct sequences of Exo70 (unpublished data). Importantly, knockdown of Exo70 did not evoke any change in cell shape or proliferation, expression of insulin receptor or Glut4, or insulin-stimulated phosphorylation of Akt (Figure 1, C-E). It should also be pointed out that it is uncertain whether total protein changes reflect fractional changes in all cells or large changes in fractions of cells.
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Insulin Controls the Localization of the Exocyst Complex
To evaluate the role of lipid rafts in coordinating the assembly of the exocyst complex, we performed a number of immunohistochemistry and fractionation experiments to assay the localization of exocyst proteins in these microdomains. Differentiated 3T3L1 adipocytes were transfected with cDNAs expressing HA-TC10 and its mutants, along with myc-Exo70 and caveolin-eGFP as a marker of lipid rafts. The cells were then stimulated with insulin, and plasma membrane sheets were prepared by a triton extraction method that leaves only lipid raft proteins on the cover glass (Watson et al., 2001). As previously reported (Watson et al., 2001; Chiang et al., 2002), both caveolin and TC10 constitutively reside in detergent-extracted sheets derived from untreated and insulin-stimulated cells. Exo70 protein was not detected in these sheets in untreated cells, but was found in sheets from cells treated with insulin. Moreover, coexpression with constitutively active TC10 (Q67L) produced the localization of Exo70 at lipid rafts even without insulin stimulation (Figure 2A). Although we are unable to detect endogenous Sec8, we transfected cells with Myc- or HA-tagged Sec8 and treated cells with or without insulin. Insulin stimulated the translocation of Sec8 to the plasma membrane. This effect of the hormone was mimicked by cotransfection of cells with constitutively active TC10 (Supplementary Figure 1).
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; Chiang et al., 2002; Kimura et al., 2002). Adipocytes were treated with or without insulin for 5 min, followed by lysis and fractionation. Caveolin1 was exclusively detected in the lipid raft-enriched fraction 4 and 5 of the gradient and was unaffected by insulin treatment, as previously shown (Chamberlain and Gould, 2002). In contrast, the transferrin receptor was exclusively found at the bottom of the gradient in fractions 10-12, where it increased in response to insulin (Chamberlain and Gould, 2002). Interestingly, Sec8 was detected in both raft (fractions 4 and 5) and nonraft fractions (10-12) in untreated cells. However, both proteins were increased only in the lipid raft fractions in cells treated with insulin (Figure 2B).
To analyze the kinetics of this effect of insulin in more detail, we performed a time course of insulin stimulation. After hormone treatment, the plasma membranes were purified and fractionated further on sucrose gradients, and the raft and nonraft fractions were pooled before immunoblotting. Although the proteins residing in the nonraft fractions did not change as a consequence of insulin stimulation, treatment of cells with insulin produced the rapid and transient translocation of both Exo70 and Sec8 into the lipid raft fraction. This effect of insulin was maximal at 5 min and declined thereafter (Figure 2C).
Glut4 Vesicles Transit through Lipid Rafts before Fusion
Glut4-containing vesicles translocate to the plasma membrane in response to insulin stimulation (Saltiel and Kahn, 2001). However, whether these vesicles are targeted to discrete sites on the plasma membrane is not known. Because the exocyst appears to assemble in large part at lipid rafts, we sought to evaluate whether Glut4 might be targeted to these microdomains early in the translocation process. Thus, to evaluate the kinetics of Glut4 translocation to the plasma membrane, we examined the time course of Glut4 translocation using the fractionation method described above and determined the concentration of Glut4 in lipid raft and nonlipid raft fractions of the plasma membrane after insulin stimulation (Figure 3A). Glut4 was rapidly translocated to rafts in response to insulin. This effect was observed in as little as 2 min and reached maximal levels 10 min after insulin stimulation. The translocation of Glut4 into raft fractions preceded the appearance of the protein in nonlipid raft fractions, where peak levels were detected 30 min after insulin stimulation. Thus, these data suggest two possible models: one in which Glut4 is initially translocated to lipid rafts, and then transits to nonraft fractions, perhaps after fusion of the vesicles, and a second in which there are two pools of Glut4, a fast pool targeted to rafts and a slower pool target to nonraft regions.
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We next examined the effect of Exo70 knockdown on the translocation of Glut4 to detergent-extracted plasma membrane sheets. Cells were electroporated with Exo70 RNAi or a scrambled oligo and then treated with or without insulin for 10 min. In scrambled oligo-transfected cells, endogenous Glut4 and caveolin costained in circular rosette structures in plasma membrane sheets, suggesting that a subpopulation of Glut4 vesicles translocate to lipid rafts after insulin stimulation (Figure 3C). Knockdown of Exo70 disrupted the colocalization of Glut4 and caveolin in rosettes, suggesting that the exocyst complex selectively tethers Glut4 to lipid rafts in the early phase of insulin signaling.
We previously reported that an Exo70 mutant containing only the N-terminal sequences (Exo70-N) inhibited insulin-stimulated glucose uptake (Inoue et al., 2003). To evaluate the mechanism of action of this mutant form of Exo70, we examined its localization in the plasma membrane. 3T3L1 adipocytes were electroporated with cDNAs encoding myc-Exo70-WT and myc-Exo70-N or a vector control and treated with or without insulin for 5 min. Subcellular localization of transfected and endogenous exocyst proteins was then evaluated by sucrose density gradient fractionation (Figure 4A). As shown above in untransfected cells, levels of Exo70, Sec8, and Glut4 were low in lipid raft fractions from unstimulated cells expressing Exo70-WT, but increased upon insulin stimulation. In contrast, the overexpression of myc-Exo70-N in adipocytes suppressed the insulin-dependent translocation of Exo70, Sec8, and Glut4 to lipid raft fractions. These data suggest that endogenous Exo70 and other components of the exocyst complex are competitively displaced from lipid rafts by overexpression of the dominant-negative form of the protein.
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). We thus screened a PDZ protein array with bacterially expressed His-tagged Sec8 as bait and identified as an interacting partner SAP97, a member of the MAGUKs (membrane-associated guanylate kinase homologues) family (Funke et al., 2005). The specificity of this interaction was evaluated in Cos-1 cells cotransfected with cDNAs encoding HA-tagged Sec8 and YFP-tagged SAP97 (Figure 5A). SAP97 was coprecipitated with full-length Sec8, but not with a mutant Sec8 lacking a PDZ-binding motif in its C-terminus (Sec8-4aa) (Figure 5A), verifying that SAP97 binds to Sec8 through the PDZ-binding motif in the C-terminus.
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SAP97 is reportedly expressed in lipid rafts in muscle cells (Folco et al., 2004). We explored the localization of SAP97 in 3T3L1 adipocytes by both immunostaining and sucrose density gradient fractionation. SAP97 clearly colocalized with caveolin in rosette structures in plasma membrane sheets (Figure 5B). Furthermore, we detected SAP97 in lipid raft fractions on sucrose density gradients (Figure 5C). There was little effect of insulin on the localization of SAP97.
To elucidate the functional significance of SAP97 as a binding partner of the exocyst protein Sec8, we knocked down SAP97 using RNAi oligo (Figure 6A). Knockdown of the SAP97 protein by 80% caused a marked reduction in the levels of both Sec8 and Exo70 in lipid rafts after insulin stimulation. Moreover, this reduction in SAP97 levels caused a blockade of the insulin-stimulated translocation of Sec8, Exo70, and Glut4 into lipid rafts, although the total amounts of these proteins in cell lysates were not altered (Figure 6B). Interestingly, the decreased content of the exocyst proteins in lipid rafts was accompanied by a significant increase of these proteins in nonlipid raft domains.
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As a further evaluation of specificity regarding the effects of knockdown of components of the exocyst complex and related proteins, we first evaluated levels of insulin receptor in plasma membrane fractions (Supplementary Figure S2). As expected, receptor levels were diminished after treatment of cells with insulin for 10 min, due to internalization of the receptor. However, knockdown of Exo70, SAP97, Sec6, or Sec8 had no effect on insulin receptor levels in this plasma membrane fraction. We also evaluated the activation of the MAP (mitogen-activated protein) kinase pathway by insulin via assay of extracellular signal-regulated kinase (ERK) phosphorylation (Supplementary Figure S3). Knockdown of this same collection of exocyst proteins had not effect on the phosphorylation of ERK in response to insulin.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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; Bickel, 2002; Muller, 2002; Saltiel and Pessin, 2002, 2003; Cohen et al., 2003a). The activated insulin receptor specifically catalyzes the tyrosine phosphorylation of certain proteins in lipid rafts, including caveolin (Kimura et al., 2002), APS (Liu et al., 2002), and Cbl (Ribon and Saltiel, 1997). Components of insulin signaling are constitutively localized in lipid rafts, including some or all of the insulin receptor (Gustavsson et al., 1999; Kimura et al., 2002), flotillin (Baumann et al., 2000; Liu et al., 2005), and TC10 (Watson et al., 2001), which is specifically activated by insulin in these microdomains due to the recruitment of the exchange factor C3G via the tyrosine phosphorylation of c-Cbl and Cbl-b (Liu et al., 2003). Disruption of lipid rafts with cholesterol-extracting drugs such as beta-cyclodextrin or by overexpression of dominant-negative forms of caveolin block the actions of insulin, without disrupting other cellular functions (Gustavsson et al., 1999; Parpal et al., 2001; Ros-Baro et al., 2001; Watson et al., 2001; Shigematsu et al., 2003). Moreover, introduction of a modification of TC10 that prevents lipid raft association blocks the activation of the G protein by insulin, as well as the stimulation of glucose uptake in fat cells (Watson et al., 2001). Targeted disruption of the gene encoding the adipose-enriched raft protein caveolin1 also produced insulin resistance, although it remains uncertain whether this resulted from decreased insulin signaling (Cohen et al., 2003b). Taken together, these data suggest that lipid rafts serve as sites for assembly of signaling complexes that play an important role in insulin action.
Why are lipid raft microdomains crucial to the downstream actions of insulin? One possible explanation is that TC10 effectors are targeted to these regions as sites for Glut4 docking and fusion. The exocyst protein Exo70 binds specifically to activated TC10 in an insulin-dependent manner (Inoue et al., 2003). This interaction results in the assembly of components of the exocyst complex, which plays a critical role in the targeting of the Glut4 vesicle to the plasma membrane, perhaps by directing the vesicle to the precise site of fusion. To understand how the exocyst complex is stabilized in these domains, we searched for Sec8 binding partners and identified SAP97, a member of the MAGUKs family of proteins. SAP97 is expressed in lipid rafts in adipocytes, suggesting that by anchoring Sec8 it might stabilize the exocyst complex in these microdomains. In this regard, knockdown of SAP97 disrupted the exocyst complex in lipid rafts and decreased insulin-stimulated translocation of Glut4 to lipid rafts, accompanied by decreased glucose uptake.
The presence of Glut4 in lipid rafts has been controversial (Bickel, 2002). Biochemical isolation and immunogold electron microscopy of plasma membranes revealed the transient localization of Glut4 immunoreactivity in lipid rafts after insulin stimulation of 3T3L1 adipocytes (Scherer et al., 1994; Gustavsson et al., 1996; Karlsson et al., 2002), although others found no evidence for Glut4 in lipid rafts (Voldstedlund et al., 1993; Kandror et al., 1995). Although this discrepancy might be the result of different methodologies, it seems possible that Glut4 initially docks and fuses at lipid rafts, due to the assembly of the exocyst in this microdomain, and then transits into nonraft domains. This idea is supported by data presented in Figure 3, in which lipid raft-association of Glut4 tended to precede the appearance of nonraft transporter. On the other hand, it is also possible that there are two populations of Glut4-containing vesicles, a rapidly docking pool that is directed to lipid rafts and another slower pool targeted to nonlipid rafts. In either case, the temporal and spatial compartmentalization of the exocyst complex to lipid raft subdomains appears to be critical to the docking and fusion of Glut4 at the cell surface.
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ACKNOWLEDGMENTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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Footnotes |
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The online version of this article contains supplemental material at MBC Online (http://www.molbiolcell.org). 
Address correspondence to: Alan R. Saltiel (saltiel{at}umich.edu).
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