To evaluate the role of TUG in the control of glucose uptake, we transfected 3T3-L1 adipocytes with synthetic siRNA duplexes. demonstrates that two different siRNA duplexes effectively deplete TUG protein. In control, mock transfected cells, and in those transfected with irrelevant siRNA duplexes, there is no effect on TUG protein amount. In contrast, the two siRNAs that target TUG each deplete the protein by ~85%, assessed 48 h after transfection. Amounts of insulin receptor β chain are unaffected. Thus, the two different siRNAs effectively and specifically deplete TUG protein in 3T3-L1 adipocytes.
Figure 1 Effect of transient, siRNA-mediated TUG depletion on glucose uptake. a. 3T3-L1 adipocytes were electroporated with synthetic siRNA duplexes as indicated. 24 or 48 h after transfection, cells were lysed and analyzed by SDS-PAGE and immunoblotting. Control (more ...)
The effect of siRNA-mediated TUG depletion on glucose uptake in 3T3-L1 adipocytes is shown in . In control mock- or luciferase siRNA- transfected cells, insulin stimulates an approximately fourfold increase in glucose uptake. In cells transfected with either of the two TUG siRNAs, basal glucose uptake is markedly increased, and no further increase is observed after insulin stimulation. In several experiments, siRNA duplex B was the slightly more effective of the two duplexes, and it may enhance glucose uptake more than siRNA duplex A, though this did not reach statistical significance. We conclude that transient depletion of TUG markedly augments glucose uptake, similar to insulin, in unstimulated 3T3-L1 adipocytes.
To determine if the enhanced glucose uptake we observed after TUG siRNA transfection is attributable to increased GLUT4 or GLUT1 protein amounts, these proteins were assayed on immunoblots of total cell lysates. As shown in , neither GLUT4 nor GLUT1 is increased in 3T3-L1 adipocytes transfected with TUG siRNA B, compared to mock transfected control cells. Indeed, GLUT4 is actually decreased, raising the possibility that TUG may coordinately regulate GLUT4 targeting and protein levels, as discussed further below. No consistent effects on GLUT1 protein amount were noted. Thus, the enhanced glucose uptake we observe cannot be attributed to increased GLUT4 or GLUT1 protein amounts. These data are consistent with the possibility that TUG depletion causes GLUT4 targeting to the plasma membrane to enhance glucose uptake in cells not treated with insulin.
To further study the effect of siRNA-mediated TUG depletion on GLUT4 subcellular localization, we performed confocal microscopy of 3T3-L1 adipocytes stably expressing a GFP-tagged GLUT4 reporter (13
). As shown in , cells transfected using a control siRNA and not treated with insulin target GLUT4 to intracellular membranes, and the typical perinuclear distribution is observed (top row). Minimal surface-exposed myc epitope tag is detected in these unstimulated cells. After insulin exposure, GLUT4 is seen in both perinuclear and plasma membrane distributions, and detection of externalized myc epitope tag demonstrates that GLUT4 has been incorporated into the plasma membrane (second row). In cells in which TUG is depleted by transfection of siRNA B, GLUT4 is observed at the cell surface even in the absence of insulin (third row). Externalized myc epitope is abundant, showing that this GLUT4 has been incorporated into the plasma membrane. The surface GLUT4 may be more punctate than in the insulin treated control cells, which have a more homogeneous pattern, yet the overall pattern is clearly distinct from that in unstimulated control cells. After insulin stimulation of the TUG siRNA-treated cells, GLUT4 is again observed at the cell surface (bottom row). Externalized myc tag is readily detected, and again demonstrates a punctate distribution. Thus, the main result is that transient, siRNA-mediated TUG depletion causes marked incorporation of GLUT4 into the plasma membrane in unstimulated 3T3-L1 adipocytes.
Figure 2 Effect of siRNA-mediated TUG depletion on GLUT4 distribution. a. 3T3-L1 adipocytes stably expressing a myc- and GFP-tagged GLUT4 protein (ref. 13) were electroporated with luciferase siRNA (control) or with siRNA duplex B (TUG siRNA), then seeded to coverslips. (more ...)
To perform more detailed analyses, we constructed 3T3-L1 cells in which TUG is stably depleted by retroviral expression of a short hairpin RNA (shRNA). This shRNA targets the same sequence as TUG siRNA duplex B, described above. We isolated a homogeneous population of infected 3T3-L1 preadipocytes by flow cytometry. These “shRNA” cells undergo normal adipose differentiation, as judged by lipid accumulation and expression of GLUT4, compared to control, uninfected 3T3-L1 cells. demonstrates that TUG is depleted by >95% in adipocytes containing the shRNA. To further control for the specificity of TUG depletion, the shRNA cells were superinfected with a retrovirus containing a shRNA-resistant form of TUG, to make “shRNA+TUG” cells. These cells were also isolated by flow sorting and undergo normal adipose conversion. shows that the reintroduced TUG protein is present in amounts that are approximately fivefold higher than that of endogenous TUG in control, uninfected 3T3-L1 adipocytes. As well, amounts of GLUT4 are similar or slightly decreased in shRNA cells, and markedly increased in shRNA+TUG cells, suggesting that GLUT4 abundance may be regulated by TUG protein levels. No large effect on GLUT1 protein amounts is observed. Thus, data from both stable and transiently transfected 3T3-L1 adipocytes suggest that TUG abundance may positively regulate GLUT4 protein amount, as examined further below.
Figure 3 Effects of stable shRNA-mediated TUG depletion on glucose uptake. a. TUG was depleted stably in 3T3-L1 cells using a shRNA-producing retrovirus to make “shRNA cells”. Wildtype, shRNA-resistant TUG was expressed stably in the shRNA cells (more ...)
We first tested the effect of stable TUG depletion on glucose uptake. shows that unstimulated 3T3-L1 adipocytes containing the shRNA have increased glucose uptake compared to control cells. This increased basal glucose uptake is nearly equal to that caused by insulin treatment of the control cells. Insulin further enhances glucose uptake in cells depleted of TUG, but only by about twofold because of the increased basal uptake. The enhanced glucose uptake in the shRNA cells is not attributable to increased GLUT1 or GLUT4 protein amounts, which are similar or slightly decreased in shRNA cells compared to control cells, as noted above (). Reintroduction of shRNA-resistant TUG rescues the effect on glucose uptake, indicating that it results from TUG depletion and is not a nonspecific effect of the shRNA. Remarkably, glucose uptake in shRNA+TUG cells is similar to that in control cells even despite the markedly increased GLUT4 abundance in shRNA+TUG cells (). This may reflect the ability of overexpressed TUG to retain GLUT4 within cells, sequestering it away from the plasma membrane (ref. 15
, ). Finally, it is notable that the variability in glucose uptake measurements is much smaller in stable cells () than in electroporated cells (), permitting the observation of a significant effect of insulin to enhance glucose uptake in the shRNA cells. Thus, both stable and transient depletion of TUG result in increased glucose uptake in unstimulated 3T3-L1 adipocytes, and in neither case is this attributable to increased amounts of GLUT4 or GLUT1 proteins.
Figure 4 Effects of shRNA-mediated TUG depletion on GLUT4 and GLUT1 distribution. a. Control, shRNA, and shRNA+TUG 3T3-L1 adipocytes were treated with insulin as indicated. Cells were homogenized and plasma membrane (PM), light microsome (LM), and heavy microsome (more ...)
To study the effect of the TUG shRNA on glucose transporter distribution, we performed subcellular fractionation of basal and insulin stimulated control, shRNA, and shRNA+TUG 3T3-L1 adipocytes. As shown in , insulin increases GLUT4 in plasma membranes of control cells, and there is a corresponding decrease of GLUT4 in light microsomes (top left panel). In shRNA cells, GLUT4 is abundant in plasma membranes, and is relatively decreased in light microsomes, both in basal and insulin treated cells (middle left panel). Some GLUT4 remains in basal light microsomes and is decreased by insulin, but this effect is less marked than in control cells and may be due to endosome recycling. In the shRNA+TUG cells, reintroduction of TUG rescues both basal GLUT4 retention in light microsomes, and insulin stimulated GLUT4 movement to the plasma membrane (bottom left panel). Therefore these effects are due to TUG depletion and not nonspecific effects of the shRNA. As predicted by data in , GLUT4 is very well excluded from plasma membranes of basal shRNA+TUG cells. Immunoblotting of the fractions to detect GLUT1 demonstrates that in control cells, insulin causes some movement of GLUT1 out of light microsomes and to the plasma membrane (top right panel), consistent with previous data (22
). TUG depletion slightly enhances GLUT1 targeting to plasma membranes in basal 3T3-L1 adipocytes. This effect is rescued by reintroduction of TUG, and is thus due to TUG depletion, yet it is much less marked than the effect on GLUT4 targeting (middle and bottom right panels). Insulin stimulates an approximately twofold further increase in plasma membrane GLUT1 in shRNA cells, and also appears to increase GLUT1 in light microsomes. Overall, depletion of TUG by a stably expressed shRNA has a large effect on GLUT4 targeting, and much smaller effect on GLUT1 targeting, in 3T3-L1 adipocytes. The main effect is marked redistribution of GLUT4 out of light microsomes and to the plasma membrane in the absence of insulin.
To quantify the effect of the TUG shRNA on GLUT4 distribution, immunoblots from several experiments were analyzed by densitometry. No large effects of TUG depletion on heavy microsomal GLUT4 were observed, thus only plasma membrane and light microsome fractions from basal and insulin stimulated cells were included in this analysis. shows that TUG depletion significantly increases GLUT4 in plasma membranes, and decreases GLUT4 in light microsomes, in unstimulated 3T3-L1 adipocytes. No significant effects were observed in insulin stimulated cells. Together with the results above, the data are consistent with the notion that TUG depletion redistributes GLUT4 from light microsomes to the plasma membrane, and that this is sufficient to increase glucose uptake in unstimulated 3T3-L1 adipocytes.
We studied the effect of a dominant inhibitory fragment of TUG, UBX-Cter, using 3T3-L1 adipocytes stably expressing this protein. Previous data suggest that this fragment prevents GLUT4 retention in an intracellular, insulin-responsive pool of GSVs within unstimulated 3T3-L1 adipocytes (15
). Thus it was anticipated that expression of UBX-Cter and RNAi-mediated TUG depletion may have similar effects. Results from glucose uptake experiments, shown in , support this prediction. The data show that UBX-Cter expression substantially increases glucose uptake in unstimulated 3T3-L1 adipocytes, such that it is indistinguishable from that of insulin treated control cells. Insulin further increases glucose uptake in cells containing UBX-Cter, but only by about twofold because of the enhanced basal uptake. As shown in , there is at most a minor increase in GLUT1 amount, and no change in GLUT4 amount, in UBX-Cter cells compared to control cells. Thus, altered glucose transporter abundance cannot account for the markedly enhanced glucose uptake observed in UBX-Cter cells, consistent with the notion that UBX-Cter may enhance glucose uptake by targeting GLUT4 to the plasma membrane.
Figure 5 Effects of UBX-Cter, a dominant negative TUG protein fragment, on glucose uptake. a. 3T3-L1 adipocytes stably expressing UBX-Cter and control cells were differentiated in multiwell plates and used for glucose uptake experiments. Counts of [3H]-2-deoxyglucose (more ...)
We further characterized the effect of the UBX-Cter fragment on GLUT4 and GLUT1 distribution by performing subcellular fractionation of 3T3-L1 adipocytes. shows that GLUT4 is markedly increased in plasma membranes prepared from unstimulated cells containing UBX-Cter (top panel). There is some further increase after insulin treatment, however this is minimal because of the abundant basal amount. In unstimulated cells, UBX-Cter decreases the amount of GLUT4 in light microsomes, suggesting that some of the increased plasma membrane GLUT4 originates from these membranes. Insulin further reduces GLUT4 in light microsomes of UBX-Cter expressing cells, yet this is a small effect that may be due to endosome recycling. Similar findings are noted for GLUT1, although the effect on plasma membrane GLUT1 is much less marked because of the abundance of GLUT1 in plasma membranes from unstimulated cells (middle panel). Expression of the UBX-Cter fragment had no large effect on heavy microsome GLUT4 or GLUT1. In , the effects on GLUT4 are quantified, based on densitometry of several experiments. These data show that UBX-Cter significantly increases GLUT4 in plasma membranes, and decreases GLUT4 in light microsomes, from unstimulated 3T3-L1 adipocytes. These results are consistent with previous microscopy data suggesting that GLUT4 and TUG colocalize in GSVs, as well as with previous kinetic data suggesting that the TUG UBX-Cter fragment prevents the basal accumulation of GLUT4 in GSVs (15
). Indeed, data presented here demonstrate a more marked effect on basal GLUT4 distribution to the plasma membrane. The flow cytometric assay used previously is better suited to kinetic measurements, and the increase in cell surface GLUT4 that was observed was likely underestimated because of the significant background fluorescences inherent in the assay. Data shown here suggest that the fragment not only blocks the retention of GSVs, but also further indicate that this is sufficient to target GLUT4 to the plasma membrane and to enhance glucose uptake in unstimulated 3T3-L1 adipocytes.
Figure 6 Effects of UBX-Cter on GLUT4 and GLUT1 distribution in 3T3-L1 adipocytes. a. Plasma membrane (PM), light microsome (LM), and heavy microsome (HM) fractions were purified from basal and insulin stimulated cells containing UBX-Cter, and from control cells. (more ...)
Confocal microscopy results are consistent with this interpretation. shows the distribution of stably expressed myc- and GFP-tagged GLUT4 in control 3T3-L1 adipocytes and in cells expressing the dominant negative UBX-Cter fragment. In control cells, insulin causes the appearance of a homogeneous rim of GLUT4 at the plasma membrane. The incorporation of GLUT4 into the plasma membrane is demonstrated by staining of externalized myc epitope tag in the intact cells (second row). In cells containing UBX-Cter, there is clear targeting of GLUT4 to the plasma membrane of unstimulated cells (third row). In these cells, the GLUT4 is incorporated into the plasma membrane, since extracellular myc tag can be detected. The plasma membrane GLUT4 is more punctate in appearance than in insulin stimulated control cells, particularly as assessed by GFP, similar to findings with TUG siRNA (). After insulin stimulation, the UBX-Cter cells have a slightly more homogeneous rim of GLUT4 at the cell surface (bottom row). As discussed below, this finding is consistent with the notion that insulin stimulates fusion of vesicles containing GLUT4 with the plasma membrane, which may also account for the twofold further increase in glucose uptake upon insulin stimulation of cells containing UBX-Cter. Overall, the data show that UBX-Cter causes targeting and incorporation of substantial GLUT4 into the plasma membrane. Together with results above, we conclude that the effect of the dominant negative TUG UBX-Cter fragment on GLUT4 is equivalent to that of RNAi-induced TUG depletion. Both cause GLUT4 targeting to the plasma membrane, and this is sufficient to augment glucose uptake in 3T3-L1 adipocytes.
Figure 7 Effect of UBX-Cter on GLUT4 distribution. Control and UBX-Cter expressing 3T3-L1 adipocytes containing a myc- and GFP-tagged GLUT4 reporter were starved, treated with or without 160 nM insulin for 15 min., then chilled to 4°C and stained to detect (more ...)
To further test if TUG controls the basal accumulation of GLUT4 in a nonendosomal, perinuclear compartment, we performed confocal microscopy of unstimulated 3T3-L1 adipocytes. Our previous data show that TUG colocalizes with GLUT4, and not with the endosomal marker TfnR, on intracellular membranes of unstimulated 3T3-L1 cells (15
). Here, we examined the distributions of GLUT4 and TfnR in the perinuclear region of cells overexpressing TUG, or expressing UBX-Cter fragment. shows that in control cells, despite the substantial overlap of GLUT4 and TfnR, membranes that contain GLUT4 and lack appreciable TfnR are readily visualized (arrowheads, first and second rows). These membranes correspond to nonendosomal vesicles containing GLUT4. When TUG is overexpressed, these GLUT4-positive, TfnR-negative membranes are much more prominent (arrowheads, third and fourth rows). This suggests expansion of a nonendosomal pool of GLUT4 in cells overexpressing TUG. By contrast, when UBX-Cter is expressed, the GLUT4-positive, TfnR-negative vesicles are essentially undetectable (fifth and sixth rows). In these cells, GLUT4 is targeted to the plasma membrane, as noted above ( and bottom left panel of ). Strikingly, the remaining intracellular GLUT4 overlaps completely with TfnR. These findings suggest that UBX-Cter mediated disruption of TUG function results in targeting of GLUT4 to endosomes. Together with data shown above, we conclude that TUG overexpression and TUG UBX-Cter expression have opposite effects. TUG overexpression enhances the normal accumulation of GLUT4 in intracellular, nonendosomal membranes, concurrent with its sequestration away from the plasma membrane. UBX-Cter expression disrupts this action, so that GLUT4 distribution is similar to that of TfnR-containing endosomes and GLUT4 is targeted to the plasma membrane.
Figure 8 Effects of TUG overexpression or UBX-Cter expression on distribution of perinuclear GLUT4. Unstimulated 3T3-L1 adipocytes containing GFP-tagged GLUT4 were starved, fixed and permeablized, and stained by indirect immunofluorescence to detect transferrin (more ...)
We hypothesized that the effects on GLUT4 targeting we observed may have secondary consequences for GLUT4 protein stability. Previous work indicates that GLUT4 is degraded mainly by lysosomes (14
). Therefore we considered that TUG-mediated retention of GLUT4 in nonendosomal, intracellular membranes may sequester GLUT4 away from lysosomes, as well as from the plasma membrane, thus prolonging GLUT4 half-life. This possibility was suggested by the decreased GLUT4 we observed after siRNA-mediated TUG depletion in 3T3-L1 adipocytes (). In this transient experiment, done using fully mature 3T3-L1 adipocytes, potential effects of TUG disruption on 3T3-L1 differentiation would not be expected to play a role. The proposed mechanism may also contribute to the increased GLUT4 observed in stable 3T3-L1 cells overexpressing TUG, as in shRNA+TUG cells ().
To test this hypothesis, we first sought to determine more definitively if siRNA-mediated TUG depletion affects GLUT4 protein abundance, independently of GLUT4 mRNA abundance. We reasoned that an effect on GLUT4 protein stability might be most dramatic in cells not exposed to insulin, because GLUT4 targeting is most affected by TUG disruption in unstimulated cells. Accordingly, cells were transfected with TUG siRNA B or with a control siRNA, then cultured in the absence of insulin or serum for 24 h before analysis of GLUT4 protein (by immunoblotting) and mRNA (by quantitative, real-time PCR). As shown in , TUG knockdown causes a dramatic decrease in GLUT4 protein abundance. This is not accompanied by a decreased GLUT4 mRNA amount, which is unchanged as shown in . The overall decrease in GLUT4 protein amount is about 60%, as assessed by densitometry of immunoblots (). Finally, control experiments show that the TUG siRNA depleted TUG mRNA and protein similarly, by about 80% ( and ). To facilitate measurement of GLUT4, the experiment shown was done using cells that stably express the GLUT4 reporter, which is about fivefold more abundant than native GLUT4 and contains a myc epitope tag. Similar results were obtained for endogenous GLUT4 mRNA and protein in 3T3-L1 adipocytes not expressing the reporter. Finally, the decrease in GLUT4 protein is slightly more marked in than in , possibly because cells in were starved for a longer period of time. In conclusion, TUG depletion can cause decreased GLUT4 protein abundance, independent of any change in GLUT4 mRNA amount.
Figure 9 Effects of siRNA-mediated TUG depletion on GLUT4 mRNA and protein abundance. 3T3-L1 adipocytes stably expressing the GLUT4 reporter were electroporated with siRNA duplexes targeting TUG (duplex B) or, as a control, luciferase (which is not present in (more ...)
The decreased abundance of GLUT4 protein after TUG depletion could result from decreased GLUT4 translation, increased GLUT4 degradation, or both. As described above, we hypothesized that loss of TUG-mediated retention may enhance GLUT4 trafficking to lysosomes, and consequently increase the rate of GLUT4 degradation. The rate of GLUT4 degradation in lysosomes can be revealed by the increase in GLUT4 abundance after chloroquine addition, since this drug inhibits GLUT4 lysosomal degradation (14
). Therefore, to test if dominant negative UBX-Cter fragment accelerates GLUT4 lysosomal degradation, 3T3-L1 adipocytes containing this fragment were treated with chloroquine for various amounts of time. As in , we reasoned that effects might be most dramatic in unstimulated cells. Therefore, to minimize insulin stimulation, cells were cultured in 0.5% fetal bovine serum before and during chloroquine addition. As shown in , chloroquine treatment modestly increases GLUT4 in control cells, and this is detected after long (14 h) incubation with this drug (top left panel). This finding is consistent with the relatively slow rate of GLUT4 turnover in unstimulated 3T3-L1 adipocytes, as described previously (23
). In cells containing UBX-Cter, chloroquine treatment much more markedly increases GLUT4 protein, and this effect is observed as soon as 4 h after its addition (top right panel). Control immunoblots demonstrate that chloroquine has no large effect on TUG itself (bottom panels). Of note, TUG is likely degraded primarily by the proteasome, since it is mostly cytosolic and contains ubiquitin-like domains. Thus, the data support the hypothesis that UBX-Cter expression accelerates GLUT4 degradation in lysosomes.
Figure 10 Effect of chloroquine on GLUT4 accumulation in 3T3-L1 cells. a. Control cells and cells containing TUG UBX-Cter were treated with chloroquine for the indicated amounts of time. Cells were lysed and equal amounts of total protein were analyzed by immunoblotting (more ...)
To further test if lysosomal GLUT4 degradation is accelerated by shRNA-mediated TUG depletion, and decelerated by TUG overexpression, similar experiments were done with chloroquine treatment of control cells, shRNA cells, and shRNA+TUG cells. shows that in control cells, chloroquine addition causes a gradual increase in GLUT4 abundance, most marked at 16 h, consistent with previously described rates of GLUT4 turnover (top left panel) (14
). In shRNA cells, chloroquine induces a larger and more rapid increase in GLUT4, suggesting that lysosomal GLUT4 degradation is more rapid in these cells (top center panel). Conversely, when TUG is overexpressed, in shRNA+TUG cells, the change in GLUT4 abundance is minimal, indicating a reduced rate of GLUT4 lysosomal degradation (top right panel). Control immunoblots show no effect of TUG depletion or overexpression on Hsc70 abundance, which is slightly decreased at the 16 h timepoint in all cases (bottom panels). Together, the data are consistent with a model in which TUG normally acts to sequester GLUT4 away from the plasma membrane in the absence of insulin, and that, in doing so, it also prevents GLUT4 trafficking to lysosomes and consequent degradation. TUG depletion or dominant negative fragment disrupt these actions to cause both enhanced glucose uptake and accelerated lysosomal GLUT4 degradation. Reintroduction of TUG in the shRNA cells not only restores highly insulin-responsive glucose uptake, but also (since the reintroduced protein is overexpressed) prolongs the half-life of GLUT4 proteins.
To study the interaction of TUG and GLUT4, we produced recombinant TUG and GLUT4 proteins. For GLUT4, the large intracellular loop between transmembrane segments 6 and 7 (TM6-7) was produced as a GST fusion, and GST alone and fused to the TM6-7 loop of GLUT1 were used as controls. Proteins were incubated together, then bound TUG was assayed by immunoblotting. As shown in , intact TUG binds specifically to the loop of GLUT4, and interacts minimally or not at all with that of GLUT1 or with GST alone. On darker exposures of the film, some interaction with the GLUT1 loop is detectable over the GST alone control, yet this is clearly lower affinity than the interaction with the GLUT4 loop. These data are consistent with previous results showing that TUG binds GLUT4, and not GLUT1, in coimmunoprecipitations using transfected cells (15
). Similar results using recombinant proteins were also obtained by X. Huang and coworkers (X. Huang, A. Rudich, N. Wijesekara, P. Bilan, J. Bogan, and A. Klip. Abstract 226, Keystone Symposium on Diabetes Mellitus, Banff, Canada, 2004).
Figure 11 Binding of recombinant TUG and GLUT4 proteins. a. GST alone, or fused to the large intracellular loop of GLUT1 or GLUT4 was produced in bacteria and immobilized on a glutathione support. Recombinant flag-tagged TUG was produced using the Roche RTS system, (more ...)
To identify an interacting region on TUG, residues 1–164 or 165–550 were produced in vitro
, and incubated with GST alone or fused to the TM6-7 loop of GLUT4. demonstrates that a TUG fragment containing residues 1–164, but not 165–550, binds directly and specifically to the GLUT4 loop. These data are consistent with previous findings that deletion of residues 1–164 inhibit the ability of TUG to efficiently retain GLUT4 within cotransfected 293 cells (Fig. 4b of ref. 15
). Previous data also show that deletion of residues 1–77 has no effect on the ability of TUG to retain GLUT4 intracellularly or to coimmunoprecipitate GLUT4 in transfected 293 cells (Figs. 2a and 4b of ref. 15
). Thus, the GLUT4 loop likely interacts directly with TUG residues 78–164.
These data strengthen and expand upon previous results, which suggested that two independent interactions may be required to mediate high affinity binding of TUG and GLUT4. Data show that forms of TUG containing truncations of the amino terminus to residue 270 are still able to partially retain GLUT4 within 293 cells, and are also able to coimmunoprecipitate GLUT4 when both proteins are overexpressed (Figs. 4b and 4c of ref. 15
). Therefore one interaction between TUG and GLUT4 is predicted to involve TUG residues 270–376. This interaction may be direct or indirect, and the required residues in GLUT4 are not known. The second interaction, characterized here, is direct and involves TUG residues 1–164 (and probably 78–164, as noted above) together with the large intracellular loop of GLUT4. Together, the data are consistent with a model in which both interactions are required for high affinity binding and efficient intracellular retention of GLUT4 by TUG.