To examine the temporal relationship between pre‐ and post‐docking events, we generated a Munc18c temperature‐sensitive mutant (Munc18c/TS) by substitution of arginine 240 with a lysine residue. At the permissive temperature (23°C), overexpression of both the wild type (Munc18c/WT) and the R240K mutant inhibited insulin‐stimulated GLUT4/IRAP vesicle translocation. However, at the non‐permissive temperature (37°C) only Munc18c/WT inhibited GLUT4/IRAP translocation whereas Munc18c/TS was without effect. Moreover, Munc18c/WT bound to syntaxin 4 at both 23 and 37°C whereas Munc18c/TS bound syntaxin 4 only at 23°C. This was due to a temperature‐dependent conformational change in Munc18c/TS, as its ability to bind syntaxin 4 and effects on GLUT4 translocation were rapidly reversible while protein expression levels remained unchanged. Furthermore, insulin stimulation of Munc18c/TS‐expressing cells at 23°C followed by temperature shift to 37°C resulted in an increased rate of GLUT4 translocation compared with cells stimulated at 37°C. To date, this is the first demonstration that the rate‐limiting step for insulin‐stimulated GLUT4 translocation is the trafficking of GLUT4 vesicles and not their fusion with the plasma membrane.
The majority of insulin‐stimulated glucose uptake in both striated muscle and adipose tissues is attributable to the presence of the insulin‐responsive glucose transporter GLUT4 (Olson and Pessin, 1996). In the basal non‐insulin stimulated state, the GLUT4 protein is localized to the perinuclear region and in both tubulovesicular elements and small intracellular vesicles scattered throughout the cell cytoplasm (Slot et al., 1991a,b). Upon addition of insulin, a series of intracellular signaling events ensues leading to the movement (trafficking), association (docking) and eventual incorporation (fusion) of the GLUT4 storage vesicles with the plasma membrane (Czech, 1995; Kandror and Pilch, 1996; Rea and James, 1997; Pessin et al., 1999). Similarly, the insulin‐responsive aminopeptidase (IRAP) has a near identical subcellular distribution to GLUT4 and displays the same pattern of insulin‐stimulated translocation (Kandror and Pilch, 1994, 1996; Kandror et al., 1994; Ross et al., 1996; Malide et al., 1997; Martin et al., 1997). Although the physiological role of IRAP translocation remains unknown, the insulin‐stimulated translocation of the GLUT4/IRAP‐containing cargo vesicles results in a large increase in the number of functional glucose transporters on the cell surface and thereby accounts for the dramatic increase in glucose uptake.
This multi‐step translocation process shares several important features with the exocytosis of synaptic vesicles during neurotransmitter release. For example, the interaction of the GLUT4 vesicle v‐SNARE protein VAMP2 with the plasma membrane t‐SNARE proteins syntaxin 4 and SNAP23 is necessary for insulin‐stimulated GLUT4 translocation (Cheatham et al., 1996; Volchuk et al., 1996; Olson et al., 1997; Tellam et al., 1997; Martin et al., 1998). In addition to these t‐ and v‐SNAREs, there are adipocyte homologs of the n‐Sec1 regulator of synaptic vesicle trafficking, Munc18b and Munc18c, of which only the Munc18c isoform binds to syntaxin 4 with high affinity (Tellam et al., 1997; Thurmond et al., 1998, 2000). Several studies have also demonstrated that increased expression of Munc18c but not Munc18b specifically inhibits insulin‐stimulated GLUT4/IRAP translocation without affecting the trafficking of other recycling proteins (Tamori et al., 1998; Thurmond et al., 1998). More recently, it has been observed that the overexpression of Munc18c resulted in the occlusion of the plasma membrane vesicle binding sites and thereby prevented GLUT4 translocation at the step of plasma membrane docking (Thurmond et al., 2000). These data are consistent with recent structural studies of the n‐Sec1 (Munc18a) homolog, which appears to function in the transition of syntaxin 1A from the closed to open conformational state (Dulubova et al., 1999; Misura et al., 2000).
Unlike synaptic vesicle exocytosis, the rate‐limiting step for insulin‐stimulated GLUT4 translocation remains unknown. Since insulin‐stimulated translocation of the GLUT4/IRAP vesicles can be subdivided into at least four overall processes (signaling, vesicle trafficking, plasma membrane docking and fusion), we proposed that the ability to regulate Munc18c function would provide a convenient tool to distinguish signaling and trafficking events from that of docking and fusion. To this end, we have generated a temperature‐sensitive Munc18c mutant based upon its sequence similarity to yeast Sly1p. In this study, we demonstrate that the Munc18c mutant (Munc18c/TS) specifically inhibits GLUT4/IRAP‐containing cargo vesicle translocation at the permissive temperature, which is rapidly reversible upon shifting to the non‐permissive temperature. Furthermore, examination of the rate of GLUT4 translocation following temperature shift demonstrates that the rate‐limiting step in insulin‐stimulated GLUT4 translocation is the trafficking of these vesicles to the plasma membrane and not docking or fusion.
Insulin‐stimulated GLUT4 translocation occurs at both 23 and 37C in 3T3L1 adipocytes
At the physiological temperature of 37°C, GLUT4 vesicles have been shown to undergo an insulin‐stimulated translocation to the plasma membrane of 3T3L1 adipocytes with a t1/2 of ∼4 min. However, this translocation is prevented when the temperature is reduced to 19°C (Robinson et al., 1992). Recently, we have observed that insulin‐stimulated GLUT4 translocation can occur at 23°C but with a substantially slower rate, t1/2 ∼30 min (Elmendorf et al., 1999). To determine the time required for the full extent of insulin‐stimulated GLUT4 translocation in 3T3L1 adipocytes incubated at 23°C to equal that of cells incubated at 37°C, we isolated plasma membrane sheets and evaluated GLUT4 translocation using an endofacial GLUT4 antibody (Figure 1). In the basal state, very little GLUT4 immunofluorescence was detected in sheets prepared from cells incubated at either 23 or 37°C (Figure 1a and g). Upon insulin stimulation, a markedly reduced rate of GLUT4 protein immunofluorescence was observed in plasma membrane sheets isolated from cells incubated at 23°C compared with those incubated at 37°C (Figure 1a–e compared with g–k). Although the rate of GLUT4 translocation in cells incubated at 23°C was less than that of cells incubated at 37°C, similar levels were reached after 90 min of insulin stimulation (Figure 1f and l). Thus, incubation of cells at 23°C with insulin for 90 min resulted in comparable amounts of GLUT4 translocation to incubation at 37°C for 30 min.
Substitution of arginine 240 for lysine results in a temperature‐sensitive Munc18c mutant protein
One useful approach to reversibly modulate a specific step in a biological process is with temperature‐sensitive mutants. Recently, a temperature‐sensitive allele in Sly1p, a yeast homolog of Munc18c, was isolated based upon its ability to allow growth at 25°C but not at 37°C (Cao et al., 1998). Sequence analysis revealed that this phenotype was the result of a single point mutation at arginine 266 that was mutated to lysine (R266K). Amino acid alignment shows that this arginine residue is highly conserved in Sly1p homologs from yeast to mammals (Figure 2). The 13 amino acid region flanking this conserved arginine displays between 38 and 62% amino acid identity with 77–92% sequence similarity amongst all the different isoforms and species. In addition, the core sequence surrounding this arginine residue is also highly conserved and can be defined by the consensus motif LΨI/LXDRXXD, where Ψ represents a hydrophobic and X any amino acid residue.
Although Sly1p functions in the trafficking of endoplasmic reticulum‐derived vesicles to the Golgi, based upon the high degree of conservation we speculated that the same mutation in Munc18c might also result in a temperature‐sensitive phenotype. We therefore co‐transfected 3T3L1 adipocytes with an enhanced green fluorescent protein (EGFP) epitope‐tagged GLUT4 (GLUT4–EGFP) expression plasmid and either the pcDNA3 empty vector, the wild‐type Munc18c (Munc18c/WT) or the R240K Munc18c (Munc18c/TS) mutant (Figure 3). As previously reported (Thurmond et al., 1998, 2000), expression of GLUT4–EGFP in 3T3L1 adipocytes results in a similar distribution to the endogenous GLUT4, being primarily localized to the perinuclear region and small vesicles throughout the cytoplasm in cells maintained at 37°C (Figure 3g). As expected, 30 min of insulin stimulation resulted in a redistribution of the GLUT4–EGFP to the cell surface, resulting in the formation of an essentially continuous rim of fluorescence (Figure 3h). Similarly, the small vesicle and perinuclear distribution of GLUT4–EGFP was identical in cells incubated at 23°C, which also display an insulin‐stimulated translocation to the plasma membrane (Figure 3a and b). Since the rate of translocation is significantly slower at 23°C compared with 37°C (Figure 1) the 23°C incubated cells were treated with insulin for 90 min. In any case, the expression of Munc18c/WT had no effect upon the basal distribution of GLUT4–EGFP but markedly inhibited the insulin‐stimulated GLUT4–EGFP translocation at both 23 and 37°C (Figure 3, compare c with d, and i with j). Like Munc18c/WT, expression of Munc18c/TS had no effect on the basal GLUT4–EGFP localization at either temperature and prevented the insulin‐stimulated translocation at 23°C (Figure 3e, f and k). Importantly, however, expression of Munc18c/TS had no effect on the insulin stimulation of GLUT4–EGFP translocation at 37°C (Figure 3l). These data suggest that the R240K single point mutation in Munc18c resulted in the production of a temperature‐sensitive Munc18c protein.
To confirm these results, we also examined the effect of Munc18c expression on another insulin‐sensitive co‐localized cargo protein, IRAP (Figure 4). Consistent with the effects of Munc18c/WT and Munc18c/TS on GLUT4–EGFP translocation, essentially identical results were obtained with the co‐expression of the EGFP epitope‐tagged IRAP protein, EGFP–IRAP. That is, expression of Munc18c/WT inhibited the insulin‐stimulated translocation of EGFP–IRAP at both 23 and 37°C (Figure 4a–d and g–j). In contrast, the Munc18c/TS mutant was capable of inhibiting insulin‐stimulated EGFP–IRAP translocation at 23 but not 37°C (Figure 4, compare e and f with k and l). Quantitation of the number of cells displaying a continuous cell surface fluorescence indicated that insulin induced the translocation of GLUT4–EGFP in 61 and 70% of the transfected cell population at 23 and 37°C, respectively (Figure 5A, bars 2 and 8). Expression of Munc18c/WT reduced the number of cells displaying GLUT4–EGFP translocation to 37 and 38% at 23 and 37°C, respectively (Figure 5A, bars 4 and 10). In contrast, expression of Munc18c/TS reduced the translocation of GLUT4–EGFP to 32% at 23°C, but there was no significant inhibition at 37°C with 64% of the cell population displaying GLUT4–EGFP translocation (Figure 5A, bars 6 and 12). Similarly in cells expressing Munc18c/WT, insulin stimulated the translocation of EGFP–IRAP in 66% of the transfected cell population at both 23 and 37°C, which was reduced to 38 and 34%, respectively (Figure 5B, bars 2, 4, 8 and 10). However, expression of Munc18c/TS only inhibited EGFP–IRAP translocation in cells incubated at 23°C and not at 37°C (Figure 5B, bars 6 and 12).
In addition to the GLUT4/IRAP‐containing vesicles, insulin can stimulate the plasma membrane translocation of other vesicular compartments containing proteins such as GLUT1, the transferrin receptor and the cation‐independent mannose‐6‐phosphate receptor, CI/MPR (James et al., 1994; Kandror and Pilch, 1996; Rea and James, 1997; Pessin et al., 1999). Therefore, to assess the specificity of the Munc18c/TS mutant we also examined the insulin‐dependent translocation of a CI/MPR–EGFP fusion protein (Figure 5C). In this case, insulin‐stimulated the translocation of CI/MPR in ∼30 and 40% of the transfected cell population at 23 and 37°C, respectively (Figure 5C, bars 2 and 8). This lower extent of translocation is consistent with previous studies demonstrating that the endogenous CI/MPR does not undergo the same degree of translocation as GLUT4 (Oka et al., 1984; Wardzala et al., 1984). More importantly, expression of neither Munc18c/WT nor Munc18c/TS had any significant effect on the extent of insulin‐stimulated CI/MPR–EGFP translocation at either temperature (Figure 5C, bars 4, 6, 10 and 12). Together, these data demonstrate that Munc18c specifically regulates the translocation of the insulin‐responsive GLUT4/IRAP storage vesicle compartments but does not affect the general endosomal trafficking systems.
The temperature‐sensitive function of Munc18c/TS is readily reversible
To determine if the negative effect of the Munc18c/TS protein on insulin‐stimulated GLUT4 translocation was a reversible process, the 3T3L1 adipocytes were shifted from 23 to 37°C in the continuous presence of insulin (Figure 6). As expected, insulin stimulation for 90 min at 23°C resulted in the translocation of GLUT4–EGFP (Figure 6a and b). Similarly, cells maintained at 23°C that were then stimulated with insulin and immediately shifted to 37°C for 30 min also resulted in a similar extent of GLUT4–EGFP translocation (Figure 6e and f). However, although expression of Munc18c/TS prevented the insulin‐stimulated translocation of GLUT4–EGFP in cells continuously maintained at 23°C, the cells that were temperature shifted displayed an equal extent of GLUT4–EGFP translocation to controls (Figure 6c, d, g and h compared with control panels a, b, e and f). In a complementary approach, we have also observed that maintenance of Munc18c/TS‐expressing cells at 37°C, followed by a temperature shift to 23°C prevented insulin‐stimulated GLUT4–EGFP translocation (data not shown). Thus, these data demonstrate that the regulatory properties of Munc18c/TS on GLUT4 translocation are fully reversible as a function of temperature within a 30 min time period.
The most likely explanation for the reversibility of Munc18c/TS function is a change in protein conformation. However, it was formally possible that the Munc18c/TS mutant was either rapidly synthesized during the 90 min stimulation period with insulin at 23°C, or rapidly degraded at 37°C. To address the issue of protein synthesis, Munc18c/TS‐expressing cells were maintained at 37°C for 2 h followed by a temperature shift to 23°C for 90 min with insulin plus cycloheximide (Figure 7A). In the absence of Munc18c/WT or Munc18c/TS protein, 59 and 51% of the transfected cells displayed GLUT4–EGFP cell surface fluorescence after a temperature shift from 37 to 23°C in the absence or presence of cycloheximide, respectively (Figure 7A, bars 2 and 8). This fluorescence was reduced to 26 and 20% by the co‐expression of the Munc18c/WT protein (Figure 7A, bars 4 and 10). Co‐expression of the Munc18c/TS protein also inhibited translocation (to 22%) both in the absence and presence of cycloheximide treatment (Figure 7A, bars 6 and 12). These data indicate that the inhibitory effect of the Munc18c/TS was reversible and was not due to the biosynthesis of new Munc18c/TS protein at the permissive temperature. Furthermore, to determine whether Munc18c/TS was specifically and rapidly degraded compared with Munc18c/WT, Flag immunoblots of whole cell detergent extracts were performed using cells expressing Flag‐Munc18c/WT and Flag‐Munc18c/TS maintained at either 23 or 37°C (Figure 7B). Although the steady‐state expression levels of the Munc18c/TS mutant were slightly lower than those of Munc18c/WT, there were no significant changes in cells maintained at either 23 or 37°C (Figure 7B, lanes 1–4). In addition, incubation with insulin had no effect on protein expression (data not shown). Thus, changes in Munc18c/TS protein levels cannot account for the temperature sensitivity or reversibility of Munc18c/TS function on GLUT4 translocation.
Another property of Munc18c is its ability to interact specifically with the plasma membrane‐bound syntaxin 4 protein (Tellam et al., 1997; Tamori et al., 1998; Thurmond et al., 1998, 2000). We therefore compared the ability of Munc18c/TS to compete for Munc18c/WT binding at 23 and 37°C (Figure 8A). In this assay system, co‐expression of syntaxin 4 with EGFP‐Munc18c induces a strong plasma membrane fluorescence and competition of this interaction results in a re‐distribution of EGFP‐Munc18c to the cytosol (Thurmond et al., 2000). In the absence of competitor protein, 72 and 77% of the transfected cells displayed cell surface fluorescence at 23 or 37°C, respectively (Figure 8A, bars 1 and 4). This fluorescence was reduced to 41 and 45% by the co‐expression of the Munc18c/WT protein (Figure 8A, bars 2 and 5). In contrast, competition by the Munc18c/TS protein only occurred at 23°C with no significant effect at 37°C (Figure 8A, bars 3 and 6). These data indicate that the Munc18c/TS protein is capable of interacting competitively with Munc18c/WT for syntaxin 4 binding at the permissive temperature but not at the non‐permissive temperature.
To examine the temperature‐dependent interaction between Munc18c/TS and syntaxin 4 more directly, we next co‐expressed Munc18c/WT or Munc18c/TS proteins with syntaxin 4 in CHO/IR cells (Figure 8B). These cells contain very low endogenous levels of Munc18c but express high levels of the transfected proteins (data not shown). Syntaxin 4 immunoblots of whole cell lysates isolated from cells incubated at 23 and 37°C demonstrated similar expression levels of the syntaxin 4 protein (data not shown). Immunoprecipitation of Flag‐Munc18c/WT and Flag‐Munc18c/TS proteins demonstrated the co‐immunoprecipitation of the syntaxin 4 protein (Figure 8B, lanes 1–4). However, incubation of the Munc18c/TS‐expressing cells at 37°C resulted in a decreased amount of syntaxin 4 protein that was immunoprecipitated with Flag‐Munc18c/TS (Figure 8B, lane 4). This difference was not due to unequal expression (data not shown) or immunoprecipitation of Flag‐Munc18c/TS at 23 and 37°C, as determined by Flag immunoblotting of the Flag immunoprecipitates (Figure 8B, lanes 3 and 4). Thus, these data demonstrate that binding interactions between syntaxin 4 and Munc18c/TS protein are temperature sensitive.
Signaling and/or GLUT4 vesicle trafficking is the rate‐limiting step in insulin‐stimulated GLUT4 translocation
We have recently demonstrated that increased Munc18c expression prevents GLUT4/IRAP vesicle translocation by inhibiting vesicle docking at the plasma membrane (Thurmond et al., 2000). Having now established the reversibility of Munc18c/TS function, we recognized that this property could be utilized to determine whether the rate‐limiting event in insulin‐stimulated GLUT4 translocation occurs at a pre‐ or post‐docking step (Figure 9). As controls, the GLUT4–EGFP‐expressing cells were initially maintained at 23 or 37°C (Figure 9A). The cells maintained at 37°C were then treated with insulin and the time course of insulin‐stimulated GLUT4–EGFP translocation was determined (Figure 9A, squares). In parallel, the cells maintained at 23°C were simultaneously shifted to 37°C and treated with insulin followed by the determination of GLUT4–EGFP translocation (Figure 9A, circles). In both cases the extent and time dependence of insulin‐stimulated GLUT4–EGFP translocation was essentially identical with a t1/2 of ∼4 min. These data demonstrate that pre‐incubation at 23°C does not result in cell context changes that alter the subsequent kinetics of GLUT4 translocation at 37°C.
Consistent with our previous results, incubation of 3T3L1 adipocytes expressing Munc18c/WT at 23°C followed by shifting to 37°C in the presence of insulin resulted in a very low level of GLUT4–EGFP translocation (Figure 9B, diamonds). In contrast, cells expressing Munc18c/TS, when shifted from 23°C in the absence of insulin to 37°C in the presence of insulin, displayed a time‐dependent translocation of GLUT4–EGFP (Figure 9B, squares). Under these conditions the t1/2 for GLUT4 translocation was ∼12 min, significantly longer than that observed in the absence of Munc18c/TS expression (Figure 9A). These data indicate that the temperature‐dependent inactivation of Munc18c/TS occurred relatively slowly and thereby decreased the apparent overall rate of GLUT4 translocation. Nevertheless, when the same population of cells expressing Munc18c/TS was pre‐incubated with insulin at 23°C and then shifted to 37°C, they underwent a significantly faster rate of GLUT4–EGFP translocation (Figure 9B, triangles). Thus, pre‐activation of the insulin signaling pathways and stimulation of GLUT4 trafficking prior to the release of docking inhibition results in an enhanced rate of GLUT4 translocation. These data strongly indicate that the rate‐limiting step for insulin‐stimulated GLUT4 translocation is either activation of insulin receptor downstream effectors or trafficking of the GLUT4 vesicles to the plasma membrane but not the docking or fusion of these compartments.
The insulin‐stimulated translocation of GLUT4/IRAP‐containing vesicles is a complex multi‐step process that is necessary for normal maintenance of glucose homeostasis (Kahn, 1992; Klip et al., 1994; Olson and Pessin, 1996). Several specific molecular events and signaling molecules have been identified that can be categorized into a general four‐step model (Figure 10). Initially, insulin binding to the insulin receptor activates the intrinsic protein kinase of the receptor β subunit, resulting in its autophosphorylation and tyrosine phosphorylation of several proximal docking proteins, most notably the IRS family of insulin receptor substrate proteins (White, 1998). In turn, this results in the association, activation and targeting of the phosphatidylinositol kinase (PI 3‐kinase) (Ruderman et al., 1990; Kelly et al., 1992; Chen et al., 1993; Folli et al., 1993). The active PI 3‐kinase can then generate phosphatidylinositol‐3,4,5‐trisphosphate, which is necessary for the stimulation of both protein kinase B (PKB) and atypical protein kinase C isoforms through activation of the phosphoinositide‐dependent protein kinases (PDK1 and PDK2) and/or through engagement of the PKB pleckstrin homology (PH) domain (Bandyopadhyay et al., 1997; Kotani et al., 1998; Vollenweider et al., 1999). Although the precise role of these events in the stimulation of GLUT4 translocation remains highly controversial, this initial transduction pathway can be referred to as ‘signaling’. Since the GLUT4/IRAP vesicles are physically separated from the plasma membrane, following insulin receptor signaling these intracellular storage compartments undergo vectorial movement towards the plasma membrane in a process we refer to as ‘trafficking’. Upon close approach to the plasma membrane, these juxtaposed vesicles then bind to specific receptor proteins (t‐SNAREs) on the plasma membrane and thus become ‘docked’. Finally, the docked vesicles are functionally incorporated into the plasma membrane through the physical mixing of the two lipid bilayers by membrane ‘fusion’ events.
Although several essential players in the trafficking, docking and fusion events have been identified, the specific molecular steps and processes that these proteins mediate have remained elusive. For example, several studies have observed that increased expression of Munc18c, but not Munc18b specifically inhibits insulin‐stimulated GLUT4/IRAP translocation without affecting the trafficking of other recycling proteins (Tamori et al., 1998; Thurmond et al., 1998). These data were interpreted as evidence for a specific repressor function of Munc18c and that insulin must therefore de‐repress this activity to allow for GLUT4 translocation. However, we have recently observed that Munc18c overexpression results in the occlusion of the plasma membrane vesicle binding sites and thereby masks the true function of Munc18c as a required positive fusogenic protein (Thurmond et al., 2000). This latter finding is consistent with the complete inhibition of target membrane fusion when the Munc18c homologs of Drosophila melanogaster (Rop), Caenorhabditis elegans (Unc) and Saccharomyces cerevisiae (Sec1) are genetically ablated or mutated (Novick and Schekman, 1979; Hosono et al., 1992; Harrison et al., 1994). Furthermore, a positive fusogenic role for another Munc18 homolog in yeast (Sly1p) has recently been demonstrated in the fusion of endoplasmic reticulum‐derived vesicles with Golgi target membranes (Cao et al., 1998). In this case, analysis of Sly1p function was made possible by the use of a temperature‐sensitive conditional allele that was fully functional at 23°C but inactive at temperatures above 29°C. At the non‐permissive temperature the vesicle membranes were fully capable of binding to the Golgi target membranes but were unable to undergo fusion. However, shifting to the permissive temperature allowed for fusion of these docked vesicles.
Given the extent of homology among this family of proteins both within and across species, we speculated that the identical mutation in Sly1p might confer a similar temperature sensitivity to Munc18c. In fact, our data directly demonstrate that substitution of arginine 240 for lysine in Munc18c generates a temperature‐sensitive Munc18c that is fully functional at 23°C but inactive at 37°C. Fortunately, these temperatures lie within the range that can be used to examine insulin‐stimulated GLUT4 translocation. However, even though the rate of insulin‐stimulated GLUT4 translocation was slowed at 23°C compared with 37°C, the extent of translocation was not significantly different (Figure 1). A similar temperature dependence has also been observed for IRAP translocation (Elmendorf et al., 1999). Importantly, the known proximal insulin receptor signaling events do not appear to display significant temperature dependence, at least between 23 and 37°C. For example, several laboratories have reported that insulin receptor autophosphorylation and IRS1 substrate phosphorylation occur normally at reduced temperature (Marshall and Olefsky, 1981; Carpentier, 1989; Heller‐Harrison et al., 1995). We have also found that the rate of insulin‐stimulated PI 3‐kinase activation, association with IRS1, in vivo phosphatidylinositol‐3,4,5‐ trisphosphate production and rate of PKB activation are also largely unaffected by reduced temperature (Elmendorf et al., 1999). Thus, the reduced rate of insulin‐stimulated GLUT4 translocation at 23°C probably does not arise due to a decrease in insulin signaling and therefore probably lies distal to these events.
We therefore hypothesized that expression of a reversible temperature‐sensitive Munc18c mutant could be used to distinguish between the GLUT4/IRAP vesicle trafficking, docking and fusion. This is based upon the ability of overexpressed Munc18c/WT to prevent GLUT4/IRAP vesicle docking by occluding the syntaxin 4 binding sites, without having any effects on insulin receptor signaling or trafficking (Figure 10). Thus by stimulating cells at the permissive temperature (23°C), we were able to activate both the signaling and trafficking pathways but prevented GLUT4/IRAP vesicle docking. Release of this blockade by temperature shift to the non‐permissive temperature (37°C) would then allow for vesicle progression to the dock and fused state. If docking and/or fusion were the rate‐limiting step, then release of the temperature block would have resulted in an identical rate of translocation in cells pre‐incubated with insulin at 23°C compared with those stimulated with insulin at 37°C. However, under these conditions the pre‐incubation with insulin resulted in an accelerated translocation process, indicating that either the trafficking or the signaling steps were rate limiting. Based upon the apparent relatively normal rates of proximal signal transduction at 23°C compared with 37°C, we conclude that the rate‐limiting step for insulin‐stimulated GLUT4/IRAP‐containing vesicle translocation is trafficking to the plasma membrane.
In summary, we report here the synthesis of a temperature‐sensitive form of Munc18c protein that specifically inhibits GLUT4/IRAP storage vesicle translocation at 23°C but not at 37°C. This mutation appears to cause a conformational change in the protein such that at the non‐permissive temperature it can no longer bind to syntaxin 4. Insulin pre‐incubation of adipocytes expressing the Munc18c/TS mutant at 23°C results in an accelerated rate of GLUT4 translocation compared with cells stimulated at 37°C, suggesting that vesicle trafficking is the rate‐limiting step in GLUT4/IRAP translocation. The availability of a reversible temperature‐sensitive mammalian Munc18c protein will now allow us to probe further into the molecular details of the SNARE complex directly involved in the GLUT4/IRAP vesicle fusion process.
Materials and methods
The polyclonal GLUT4, syntaxin 4 and Munc18c antibodies were obtained as described previously (Olson et al., 1997). Rabbit polyclonal IRAP antibody was kindly provided by Dr Steven Waters (Metabolex, Inc., Hayward, CA). Monoclonal antibodies to the Flag epitope (M2 and M5) and cycloheximide were purchased from Sigma (St Louis, MO). Vectashield was obtained from Vector Laboratories (Burlingame, CA). Mini‐prep DNA and DNA agarose extraction kits were purchased from Qiagen (Santa Clarita, CA). Other chemicals were reagent grade or the best quality commercially available.
The EGFP–Flag‐Munc18c, GLUT4–EGFP and EGFP–IRAP constructs were prepared as described previously (Thurmond et al., 1998). The name of the EGFP construct denotes the position of the EGFP as either amino (EGFP–IRAP) or carboxyl (GLUT4–EGFP) terminal fusions, respectively. The full‐length syntaxin 4 cDNA was obtained from Dr Richard Scheller (Stanford University) and PCR primers directed against the 5′ and 3′ UTRs were used to amplify the DNA for subcloning into the EcoRI and XhoI sites of pcDNA3 (Invitrogen). The pcDNA3.1‐Flag‐Munc18c construct described previously (Thurmond et al., 1998) was modified to include an N‐terminal Kozak sequence and start codon upstream of the Flag epitope tag to ensure a high level of expression. Synthetic complementary oligonucleotides of the following sequence were inserted into the EcoRI and SacII restriction sites to create the in‐frame Kozak/start sequence/Flag tag: 5′‐AATTCATGGATTATAAAGATGATGATGATAAAGCCGC‐3′. The resulting cDNA was inserted into the EcoRI and XhoI sites of pGEX4T‐1 so that the 106 bp region between the StuI and NdeI sites in the Munc18c/WT cDNA could be replaced with the same region containing the R240K temperature‐sensitive (TS) point mutation. The mutated region was made by annealing synthetic complementary oligonucleotides. The mutated pGEX4T‐1‐Munc18c cDNA was excised and inserted into the pcDNA3.1 vector, creating pcDNA3‐Munc18c/TS. The pcDNA3‐Munc18c/WT and pcDNA3‐Munc18c/TS plasmids were sequenced to verify identical construction and the presence of the R240K point mutation. The full‐length cDNA encoding the cation‐independent mannose‐6‐phosphate/IGF‐II receptor was obtained from Dr Richard Roth (Stanford University, Stanford, CA) in the pECE vector. To create the CI/MPR‐EGFP construct, the entire cDNA (8.4 kb) was excised by partial digestion with EcoRI and inserted upstream of the EGFP cDNA using the EcoRI site of pEGFP‐N3 (Clontech). The CI/MPR stop codon and 3′ UTR were removed by digestion with NdeI and SalI, which also removed the transmembrane domain. The transmembrane domain DNA fragment was then synthesized by PCR for direct reinsertion into the NdeI–SalI sites, and all junctions of the construct were sequenced to verify continuity and frame.
Cell culture and transient transfection
3T3L1 preadipocytes were purchased from the American Type Tissue Culture repository, differentiated into adipocytes and transfected by electroporation as described previously (Thurmond et al., 1998). As indicated in the figure legends, co‐transfected experiments were performed using 50 μg of EGFP‐tagged plasmid DNA plus 50–200 μg of additional plasmid DNA for analysis of EGFP fluorescence (see figure legends). Following electroporation, the cells were allowed to adhere to cover slips in 35‐cm tissue culture dishes for 18 h and were then serum starved for 2 h prior to temperature studies and stimulation with 100 nM insulin. Temperature studies were carried out in Boekel incubators supplied with 8% CO2 in a 4°C cold room. Chinese hamster ovary cells expressing the human insulin receptor (CHO/IR) were obtained as previously described (Waters et al., 1995). CHO/IR cells were incubated in Minimal Eagle's Medium (MEM) supplemented with 10% fetal bovine serum at 37°C and 5% CO2. Fully confluent CHO/IR cells were transiently transfected by electroporation (0.34 kV and 960 μF) with 10–40 μg plasmid DNA per cuvette as described previously (Yamauchi and Pessin, 1994). Following electroporation, the cells were allowed to adhere to 10‐cm tissue culture dishes for 40–48 h at 37°C and then shifted to either the permissive or non‐permissive temperature for 4 h.
Plasma membrane sheet assay and confocal microscopy
Control and insulin‐stimulated adipocytes were washed with ice‐cold phosphate‐buffered saline (PBS) and plasma membrane sheets were prepared as described previously (Robinson et al., 1992). Cells were treated with 0.55 mg/ml poly‐l‐lysine followed by three washes with hypotonic buffer (23 mM KCl, 10 mM HEPES pH 7.5, 1.7 mM MgCl2, 1 mM EGTA). The cells were placed in sonication buffer (3× hypotonic buffer plus 100 μM PMSF and 1 mM dithiothreitol) and sonicated with a Fisher probe membrane disrupter. Plasma membrane sheets were washed three times with sonication buffer and fixed in 4% paraformaldehyde for 20 min on ice. The fixed plasma membrane sheets were quenched for 15 min at room temperature in 100 mM glycine. The isolated plasma membrane sheets were washed and blocked with 5% donkey serum for 1 h at 37°C followed by incubation with GLUT4 antibody (1:100 dilution of antisera) for 30 min at room temperature. Plasma membrane sheets were then washed three times with PBS, incubated with Texas Red‐conjugated donkey anti‐rabbit secondary antibody for 30 min at room temperature, washed three times with PBS, overlayed with a drop of Vectashield and visualized by confocal fluorescence microscopy using a Bio‐Rad MRC 600 laser confocal microscope.
Co‐immunoprecipitation and immunoblotting
Whole cell detergent extracts were prepared by solubilization in an NP‐40 lysis buffer (25 mM Tris pH 7.4, 1% NP‐40, 10% glycerol, 50 mM sodium fluoride, 10 mM sodium pyrophosphate, 137 mM sodium chloride, 1 mM sodium vanadate, 1 mM PMSF, 10 μg/ml aprotinin, 1 μg/ml pepstatin, 5 μg/ml leupeptin) for 10 min at 4°C. Insoluble material was separated from the soluble extract by microcentrifugation for 10 min at 4°C. Whole cell lysate (2–3 mg) was combined with 4.5 μg of Flag (M2) antibody for 2 h at 4°C followed by a second incubation with protein G–Sepharose for 1.5 h. The resultant immunoprecipitates were subjected to electrophoresis on 10% SDS–PAGE followed by transfer to PVDF and immunoblotting as described previously (Thurmond et al., 1998).
We wish to thank Drs Richard Roth, Richard Scheller and Steven Waters for providing the cDNAs for CI/M6PR, syntaxin 4 and IRAP, and for the IRAP antibody, respectively. This work was supported by research grants DK33823 and DK25925 from the National Institutes of Health. D.C.T. was supported by a postdoctoral fellowship DK09813 from the National Institutes of Health.
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