GSVs Contain Cell Surface–labeled GLUT4
To facilitate the study of GLUT4 trafficking, we used a CHO cell line stably expressing a version of GLUT4 containing a c-myc epitope (CHO/G4myc) in the first exofacial domain (Kanai et al., 1993
). The GSVs from these cells were isolated by velocity sedimentation as previously described (Herman et al., 1994
), using a modification of a procedure for the isolation of synaptic vesicles (Clift-O'Grady et al., 1990
). Western blot analysis of fractions from the glycerol velocity gradient revealed that the epitope-tagged version of GLUT4 was present within two membrane populations: a class of slowly sedimenting GSVs and a pool of rapidly sedimenting membranes corresponding to endosomes and plasma membrane (Fig. ). The sedimentation characteristics of the GSVs containing the epitope-tagged transporter were indistinguishable from those previously described for GSVs containing untagged transporter (Herman et al., 1994
Figure 1 Characterization of GLUT4 small vesicles (GSVs) by velocity sedimentation analysis in CHO cells stably transfected with myc epitope– tagged GLUT4. A postnuclear supernatant from CHO/ G4myc cells was layered onto a 5–25% glycerol gradient (more ...)
To further characterize the nature of the GSVs, we analyzed the gradient fractions by Western blotting using anti-rab4, anti-rab5, and anti-TGN38 antibodies (Fig. ). Neither rab5 nor TGN38 colocalized with GLUT4 in the region of the GSVs, whereas both colocalized with GLUT4 in the region of the rapidly sedimenting membranes. There is a more slowly sedimenting membrane fraction that contains TGN38, but the peak does not coincide with that of the GSVs. Rab4 is found in the region of the GSVs, but is not enriched in those fractions, and is also found colocalizing with GLUT4 in the rapidly sedimenting peak.
To determine if GSVs in CHO cells are formed along the endocytic pathway, we labeled the glucose transporter on the cell surface, allowed internalization of the label, and then analyzed targeting to intracellular compartments. CHO/G4myc cells were incubated for 30 min at 37°C with a radioiodinated monoclonal antibody directed against the c-myc epitope (mAb 9E10). Myc epitope–tagged GLUT4 labeled at the surface was targeted to GSVs as well as to endosomal/plasma membrane fractions (Fig. A) in a manner that is analogous to the distribution of the transporter at steady-state (Fig. ), suggesting that the binding of exogenous antibody does not detectably alter the distribution of GLUT4. Free ligand was also recovered at the top of the gradient (Fig. A, right). Uptake of the anti-myc antibody into CHO cells not transfected with GLUT4 was negligible, showing that there was no significant internalization of antibody by fluid phase endocytosis (data not shown).
Figure 2 Internalization of surface-labeled myc epitope– tagged GLUT4. (A) CHO/ G4myc cells were incubated with radioiodinated anti-myc mAb 9E10 or human transferrin for 30 min at 37°C. Cells were homogenized and a postnuclear supernatant was (more ...)
In parallel, CHO/G4myc cells were incubated with radioiodinated human transferrin. The majority of the membrane-bound labeled transferrin was recovered in large endosomal/plasma membrane fractions and did not accumulate within a homogeneous population of small vesicles (Fig. A
). Note that these gradient conditions do not resolve sorting from recycling endosomes. In contrast to our observations with PC12 cells (Herman et al., 1994
), some iodinated transferrin was distributed at low levels throughout the gradient fractions. When cells were incubated with 50-fold excess of cold transferrin in the presence of radioiodinated transferrin, uptake of 125
I was nearly undetectable indicating the specificity of the transferrin internalization (data not shown).
The time-course for the delivery of surface-labeled GLUT4 to GSVs was assessed by incubating cells in the presence of 125I-9E10 for various periods of time and quantifying the GSV-associated radioactivity. After a 5-min lag, surface-labeled GLUT4 began appearing at detectable levels in the GSVs (Fig. B).
Endocytosis of surface-labeled GLUT4 was assayed at both 15° and 37°C (Fig. C). CHO/G4myc cells were incubated with radioiodinated mAb 9E10 on ice, washed, and then shifted to 15° or 37°C for various periods of time. After warming, surface-bound antibody was removed by acid stripping. The fraction of acid-resistant, cell-associated radioactivity was then plotted against time. As expected, endocytosis of GLUT4 into an acid-resistant compartment was slowed at 15°C but not blocked. Morphological analysis by immunofluorescence microscopy confirmed the accumulation of GLUT4 within an acid-resistant endosomal compartment at 15°C (see below).
GSV Formation Is Reversibly Blocked at 15°C
In an effort to dissect the nature of this pathway, we sought to identify whether there were any temperature-sensitive steps in the formation of GSVs. Previous studies have shown that at temperatures <10°C, internalization from the plasma membrane is inhibited (Marsh and Helenius, 1980
; Weigel and Oka, 1982
; Hopkins and Trowbridge, 1983
; Iacopetta and Morgan, 1983
; Steinman et al., 1983
; Trowbridge et al., 1993
). At 15°C, budding of clathrin-coated pits from the plasma membrane takes place (Schmid and Smythe, 1991
). At temperatures between 15° and 22°C, endocytosis and recycling is slowed, while transfer to the degradative pathway appears to be blocked (Dunn et al., 1980
; Weigel and Oka, 1982
; Hopkins and Trowbridge, 1983
; Iacopetta and Morgan, 1983
; Harding and Unanue, 1990
; Trowbridge et al., 1993
). Formation of synaptic vesicles from an endosomal precursor in neuroendocrine PC12 cells is completely inhibited at 15°C (Desnos et al., 1995
We examined whether GSVs could be formed at 15°C. Surface-labeled GLUT4 was internalized into endosomes but was not detectable in the GSVs (Fig. A). Even after prolonged incubation of up to 3 h at 15°C (data not shown), no labeling of GSVs was detected. Analysis of the steady-state distribution of GLUT4 by immunoblotting demonstrated that incubation at 15°C resulted in the depletion of GSVs as well, suggesting that while formation of GSVs was blocked, fusion with a target compartment could still occur (Fig. B).
Figure 3 Reversible inhibition of GSV formation at 15°C. Cells were incubated with radioiodinated anti-myc antibodies for 40 min at 37°C or 80 min at 15°C, washed, homogenized, and then fractionated as above. Fractions were counted on (more ...)
The temperature block was reversible since when cells were labeled for 80 min at 15°C, washed to remove free antibody, and then shifted to 37°C, GSV formation was again observed (Fig. C). A time-course of this recovery of GSV formation from the precursor compartment labeled at 15°C showed a half-time of vesicle formation of ~8 min with an undetectable lag time (Fig. D). These experiments suggest that the GSVs are forming from an endosomal precursor and that exit of GLUT4 out of this endosomal compartment could be blocked at low temperature.
Trafficking of Internalized GLUT4 and Transferrin Receptor Is by Distinct Pathways
Since 125I-transferrin did not accumulate in a homogeneous population of small vesicles, it was difficult to compare the effects of the 15°C block on transferrin receptor trafficking versus GLUT4 trafficking using radioactive labeling techniques. Instead, immunofluorescence microscopy was used to analyze compartments reached by GLUT4 or transferrin after internalization at 37° or 15°C.
CHO/G4myc cells were incubated with either anti-myc mAb or Texas red–coupled human transferrin at 37° or 15°C and processed for laser scanning confocal immunofluorescence microscopy. After a 30-min incubation at 37°C, transferrin was found to be distributed in punctate structures throughout the cytoplasm and in the juxtanuclear area (Fig. , A
). Most of the label was concentrated in the perinuclear structures consistent with earlier reports (Trowbridge et al., 1993
; Gruenberg and Maxfield, 1995
) describing the trafficking of transferrin from peripheral sorting endosomes to juxtanuclear recycling endosomes. When cells were incubated with Texas red–coupled transferrin in the presence of a 50-fold excess of unlabeled, iron-loaded transferrin no internalized fluorescein-treated transferrin was detected (data not shown). Furthermore, when cells were subjected to a prolonged incubation (150 min) at 15°C with Texas red–coupled transferrin, no change from the pattern observed at 37°C was seen, and both peripheral and perinuclear structures were again identified (Fig. , E
Uptake of anti-myc mAb at 37°C for 30 min resulted in a pattern similar to that of transferrin internalized under the same conditions, demonstrating that GLUT4 trafficked through heterogeneously sized peripheral and perinuclear structures (Fig. , C and D). However, when cells internalized anti-myc antibody at 15°C, the labeling of perinuclear structures could no longer be seen. Instead, labeled GLUT4 accumulated in a population of large peripheral endosomes (Fig. , G and H). Many of these structures appeared to be localized very close to the plasma membrane. Acid stripping of cells with 0.5 M acetic acid after allowing antibody internalization did not alter the labeling of the peripheral structures, suggesting that they do not communicate with the cell surface. As a control, the steady-state distribution of GLUT4 was determined in cells that were preincubated at 15°C. Large peripheral structures were predominately labeled, with a diminution in labeling of the perinuclear structures (data not shown), indicating that antibody internalization did not significantly perturb GLUT4 trafficking. Thus, although incubation at 15°C appears not to arrest transferrin trafficking, the reduced temperature limits the distribution of GLUT4 to large, peripheral endosomal compartments.
Formation of perinuclear structures containing GLUT4 could be restored by shifting cells, previously fluorescently labeled at 15°, to 37°C. Cells were incubated with either Texas red–coupled transferrin or anti-myc mAb for 150 min at 15°C, washed extensively on ice, and then chased in label-free media at 37°C. After a 10-min chase at 37°C, GLUT4 label was again detected in juxtanuclear structures as well as in heterogeneously sized cytoplasmic structures (Fig. , K and L). By 30 min, most of the label was localized to juxtanuclear structures (data not shown), and resembled the pattern seen after uptake of antibody at 37°C alone. Large, peripheral endosomes could no longer be easily identified. In the case of transferrin, after 10 min at 37°C, most of the internalized transferrin was localized to juxtanuclear structures (Fig. , I and J). By 30 min, staining was almost undetectable suggesting that most of the transferrin had recycled back to the surface and was released into the media (data not shown), as has been demonstrated earlier.
These experiments suggest that GLUT4 traffics between two separate populations of endosomes: large peripheral endosomes and perinuclear endosomes. GLUT4 movement between these two compartments appears to be directional, with traffic from peripheral to perinuclear endosomes blocked at 15°C.
The large peripheral GLUT4-containing compartment observed at 15°C was further characterized by double- labeling immunofluorescence assays. Cells were incubated at 15°C for 2.5 h with both Texas red–coupled transferrin and fluoresceinated anti-myc antibody. Cells were then either processed immediately (Fig. , A–F) for confocal microscopy or shifted to 37°C for 10 min in label-free media (Fig. , G–I), and then processed. At 15°C, large peripheral compartments containing GLUT4 but not transferrin were identified. The perinuclear structures containing transferrin were less apparent in these images because of the plane of sectioning, which was chosen to optimize the visualization of the GLUT4 structures. After shifting to 37°C, GLUT4 and TfR did appear to be colocalized to some extent in the perinuclear region, although the possibility of distinct GLUT4- and transferrin-containing compartments in very close apposition is not eliminated. In the periphery, many structures enriched for GLUT4 but not for the TfR were apparent, suggesting that GLUT4 sorts away from the TfR at 37°C as well. This latter pattern (Fig. , G–I) is identical to that seen in cells which have been incubated with both labels at 37°C for 30 min, without a prior incubation at 15°C (data not shown).
Figure 5 Double-label immunofluorescence with internalized GLUT4 and transferrin. Cells were incubated with both Texas red–coupled transferrin and fluoresceinated 9E10 at 15°C for 2.5 h, and then either processed immediately for microscopy (more ...)
Exit of GLUT4 from Peripheral Endosomes and Formation of GSVs Are Inhibited by Hypertonic Sucrose or Cytosol Acidification
The above experiments suggested that sorting mechanisms are involved in the traffic of GLUT4 and TfR in CHO cells. Previous studies have noted the presence of clathrin and COP-related coat proteins on endosomal membranes (Gruenberg and Maxfield, 1995
; Whitney et al., 1995
; Aniento et al., 1996
; Stoorvogel et al., 1996
), and suggest these proteins may play a role in the sorting of membrane proteins. In addition, subjecting cells to either incubation in media containing sucrose (Daukas and Zigmond, 1985
) or cytosol acidification with acetic acid (Davoust et al., 1987
; Sandvig et al., 1987
) has been shown to inhibit clathrin-mediated endocytosis by interfering with clathrin– adaptor interactions (Hansen et al., 1993
), or by altering the structure of clathrin itself (Heuser, 1989
; Heuser and Anderson, 1989
; Hansen et al., 1993
). We tested the effect of sucrose and cytosol acidification on GLUT4 trafficking from peripheral endosomes and on GSV formation.
Trafficking of GLUT4 out of the peripheral endosomes was again assessed by immunofluorescence microscopy. Peripheral endosomes were labeled by incubating cells with anti-myc mAb for 150 min at 15°C. Cells were then chilled on ice, washed extensively, and rewarmed in the presence or absence of 0.45 M sucrose for 10 or 30 min at 37°C. In the absence of sucrose, GLUT4 is detected in juxtanuclear structures as well as peripheral endosomes as noted above (Fig. , K and L). However, after 10 (Fig. , A and B) or 30 min (data not shown) in the presence of sucrose, no labeling of juxtanuclear endosomes was detected. Instead, labeled GLUT4 was restricted to the large peripheral endosomes and the plasma membrane. When TfR was analyzed under the same conditions, no obvious differences between the sucrose-treated and untreated cells could be detected (Figs. , I and J; and 6, C and D). By 30 min, nearly all of the internalized transferrin had exited the cells despite the presence of sucrose (data not shown), indicating that the presence of sucrose had not nonspecifically blocked all vesicular traffic.
Figure 6 Effect of hypertonic sucrose and cytosol acidification on the endocytic trafficking of GLUT4 and transferrin. Cells were incubated with either anti-myc mAb 9E10 (A, B, E, and F) or Texas red–coupled transferrin (C and D) for 2.5 h at 15°C, (more ...)
Parallel experiments were performed to test the effect of cytosol acidification on GLUT4 distribution. Peripheral endosomes were labeled as above at 15°C, cells were then incubated with acidified media containing 10 mM acetic acid, pH 5.0, to acidify the cytosol, and then shifted to 37°C for 10 or 30 min in the acidified media. As with sucrose, labeling of juxtanuclear structures was not evident (Fig. , E and F). Most of the label appeared to be restricted to the large peripheral endosomes.
If radioactively labeled GSVs form from the peripheral endosomes identified by the internalization of fluorescent label at 15°C, treatment with either hypertonic medium or cytosol acidification should also inhibit the biogenesis of GSVs. GSV formation was assayed using velocity sedimentation, after cells were labeled with radioiodinated anti-myc mAb for 80 min at 15°C, washed to remove free antibody, and then shifted to 37°C in the presence or absence of sucrose or acetic acid. Both sucrose (Fig. A
) and acetic acid (Fig. B
) completely prevented the appearance of GSVs upon rewarming the cells. Since reducing the pH can have pleiotropic effects on the cell, for a control experiment we tested the ability of GSVs to form in the presence of HCl, pH 5, which does not alter the cytosolic pH (Hansen et al., 1993
), and observed that GSV formation occurred at levels comparable to those seen in untreated cells (data not shown).
Newly Formed GSVs Are Dynamic
We next examined the fate of newly formed GSVs. To do so, we labeled GSVs, then measured their rate of disappearance under conditions that prevented additional GSV formation. CHO/G4myc cells were incubated with radioiodinated anti-myc mAb for 80 min at 15°C to label precursor endosomes, washed to remove free antibody, and then warmed for 30 min at 37°C to allow formation of labeled GSVs. Cells were then shifted to conditions that would block further GSV formation: to 15°C or to media containing hypertonic sucrose at 37°C. The amount of GSV-associated radioactivity was determined over time. In the presence of hypertonic medium containing sucrose, the GSVs rapidly disappeared (Fig. A). As a control, when cells containing labeled GSVs were incubated in media without sucrose for 30 min at 37°C, the amount of GSV-associated radioactivity was stable and unchanged (data not shown). Vesicle disappearance was also apparent during the 15°C incubation (Fig. B). When the steady-state distribution of GLUT4 was analyzed by Western blotting after incubating the cells either at 15°C (Fig. B), or with hypertonic medium (data not shown), disappearance of GSVs was also observed.
Figure 8 Redistribution of GLUT4 from GSVs to plasma membrane after inhibition of GSV formation. GSVs were labeled by incubating cells with radioiodinated anti-myc mAb for 80 min at 15°C, and then shifting cells to 37°C for 30 min. The (more ...)
We then examined the distribution of GLUT4 under these conditions that inhibit formation of GSVs but allow for their disappearance. Specifically, we compared cell surface levels of GLUT4 on control versus treated cells (Fig. C). Cells were incubated for 30 min either at 37°C in the presence or absence of hypertonic media, or at 15°C in regular media. Control and treated cells were then fixed and incubated with radioiodinated anti-myc antibody to determine the level of antibody binding to the cell surface. After 30 min in hypertonic media, the level of cell surface GLUT4 was increased more than fivefold as compared to untreated cells. After 30 min at 15°C, a threefold increase in cell surface GLUT4 was observed. These results are consistent with a redistribution of GLUT4 from GSVs to the plasma membrane. The large increase in cell surface GLUT4 seen with sucrose treatment suggests that endocytosis of GLUT4 from the surface is also efficiently blocked by sucrose. Less pronounced redistribution is seen at 15°C, consistent with our observation that at 15°C, GLUT4 can be reinternalized, albeit at a slowed rate, into peripheral endosomes. These experiments do not address whether GSVs fuse directly with the plasma membrane or must first fuse with an endosomal intermediate that subsequently delivers GLUT4 to the plasma membrane.