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Single point mutations result in the miss-sorting of Glut4 to a novel membrane compartment associated with stress granule proteins.

Song X, Lichti CF, Townsend RR, Mueckler M - PLoS ONE (2013)

Bottom Line: Insulin increases cellular glucose uptake and metabolism in the postprandial state by acutely stimulating the translocation of the Glut4 glucose transporter from intracellular membrane compartments to the cell surface in muscle and fat cells.The intracellular targeting of Glut4 is dictated by specific structural motifs within cytoplasmic domains of the transporter.We suggest that the IRM is critical for an early step in the sorting of Glut4 to insulin-responsive subcellular membrane compartments and that IRM mutants are miss-targeted to relatively large, amorphous membrane vesicles that may be involved in a degradation pathway for miss-targeted or miss-folded proteins or represent a transitional membrane compartment that Glut4 traverses en route to insulin responsive storage compartments.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology and Physiology, Washington University School of Medicine, St Louis, Missouri, United States of America.

ABSTRACT
Insulin increases cellular glucose uptake and metabolism in the postprandial state by acutely stimulating the translocation of the Glut4 glucose transporter from intracellular membrane compartments to the cell surface in muscle and fat cells. The intracellular targeting of Glut4 is dictated by specific structural motifs within cytoplasmic domains of the transporter. We demonstrate that two leucine residues at the extreme C-terminus of Glut4 are critical components of a motif (IRM, insulin responsive motif) involved in the sorting of the transporter to insulin responsive vesicles in 3T3L1 adipocytes. Light microscopy, immunogold electron microscopy, subcellular fractionation, and sedimentation analysis indicate that mutations in the IRM cause the aberrant targeting of Glut4 to large dispersed membrane vesicles that are not insulin responsive. Proteomic characterization of rapidly and slowly sedimenting membrane vesicles (RSVs and SSVs) that were highly enriched by immunoadsorption for either wild-type Glut4 or an IRM mutant revealed that the major vesicle fraction containing the mutant transporter (IRM-RSVs) possessed a relatively small and highly distinct protein population that was enriched for proteins associated with stress granules. We suggest that the IRM is critical for an early step in the sorting of Glut4 to insulin-responsive subcellular membrane compartments and that IRM mutants are miss-targeted to relatively large, amorphous membrane vesicles that may be involved in a degradation pathway for miss-targeted or miss-folded proteins or represent a transitional membrane compartment that Glut4 traverses en route to insulin responsive storage compartments.

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L500 and L503 are critical for the targeting of Glut4 to GSVs in the basal state.After co-expression of wild-type Glut4/eGFP and the various mutants within the IRM region of Glut4/Dsred by recombinant adenovirus infection, adipocytes were serum starved for 2 hours, washed with cold PBS, fixed with 4% paraformaldehyde, and then subjected to confocal microscopy analysis. The red color on the left panels represents mutants of Glut4/Dsred, the green color in the middle panels represents wild type Glut4/eGFP, and the yellow color in the right panels represents the colocalization of Glut4/eGFP and mutated Glut4/Dsred. The images were taken approximately through the middle of the cells and are representative of 4–5 independent experiments. The scale bar represents 10 μm.
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pone-0068516-g002: L500 and L503 are critical for the targeting of Glut4 to GSVs in the basal state.After co-expression of wild-type Glut4/eGFP and the various mutants within the IRM region of Glut4/Dsred by recombinant adenovirus infection, adipocytes were serum starved for 2 hours, washed with cold PBS, fixed with 4% paraformaldehyde, and then subjected to confocal microscopy analysis. The red color on the left panels represents mutants of Glut4/Dsred, the green color in the middle panels represents wild type Glut4/eGFP, and the yellow color in the right panels represents the colocalization of Glut4/eGFP and mutated Glut4/Dsred. The images were taken approximately through the middle of the cells and are representative of 4–5 independent experiments. The scale bar represents 10 μm.

Mentions: We previously noted the presence of sequence similarity between the extreme cytoplasmic C- terminal tail of Glut4 and a region within the amino terminus of the insulin responsive amino peptidase (IRAP), two membrane proteins that appear to share a major portion of their intracellular trafficking pathways in adipocytes (see Figure 1a). Two different sets of mutations within the IRM sequence motif (LXXLXPDEXD) were reported to result in aberrant subcellular targeting of Glut4 in the basal state and completely abolished its insulin-stimulated redistribution to the plasma membrane [34]. The possibility remained, however, that we had inadvertently introduced an unrelated and previously undefined dominant trafficking motif at the carboxy terminus of Glut4 in the two IRM mutants that were analyzed. We therefore examined the trafficking of 6 different single and double point mutations within the IRM sequence involving L500, L503, and P505 (see Figure 1b). The red fluorescent protein-tagged mutants were co-expressed in 3T3L1 adipocytes with green fluorescent protein-tagged wild type Glut4 in order to directly assess co-localization of the two different molecules within individual cells. We have previously demonstrated that wild type Glut4 targets indistinguishably whether it is tagged at its carboxyl-terminus with GFP or RFP, and that the tagged fusion proteins target very similarly to endogenous IRAP in 3T3L1 adipocytes [34]. Figure 2 shows that all 5 of the mutants in which either L500 and/or L503 were changed to alanine residues displayed aberrant targeting in the basal state compared to the wild type control, whereas the P505 mutant exhibited targeting that was indistinguishable from the wild type control under these conditions. Likewise, insulin-stimulated translocation of Glut4 to the cell periphery (representing presumptive plasma membrane insertion) was abolished in all 5 of the L500 and/or L503 mutants but remained intact in the single P505 point mutant (Figure 3). As observed previously, a much larger fraction of the aberrantly targeted mutants was present in large, dispersed, amorphous vesicles compared to the tagged wild-type Glut4, which was largely observed in small, diffuse, punctate structures and in a perinuclear compartment. Some co-localization between the mutants and wild-type transporters was observed in the perinuclear area.


Single point mutations result in the miss-sorting of Glut4 to a novel membrane compartment associated with stress granule proteins.

Song X, Lichti CF, Townsend RR, Mueckler M - PLoS ONE (2013)

L500 and L503 are critical for the targeting of Glut4 to GSVs in the basal state.After co-expression of wild-type Glut4/eGFP and the various mutants within the IRM region of Glut4/Dsred by recombinant adenovirus infection, adipocytes were serum starved for 2 hours, washed with cold PBS, fixed with 4% paraformaldehyde, and then subjected to confocal microscopy analysis. The red color on the left panels represents mutants of Glut4/Dsred, the green color in the middle panels represents wild type Glut4/eGFP, and the yellow color in the right panels represents the colocalization of Glut4/eGFP and mutated Glut4/Dsred. The images were taken approximately through the middle of the cells and are representative of 4–5 independent experiments. The scale bar represents 10 μm.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3713040&req=5

pone-0068516-g002: L500 and L503 are critical for the targeting of Glut4 to GSVs in the basal state.After co-expression of wild-type Glut4/eGFP and the various mutants within the IRM region of Glut4/Dsred by recombinant adenovirus infection, adipocytes were serum starved for 2 hours, washed with cold PBS, fixed with 4% paraformaldehyde, and then subjected to confocal microscopy analysis. The red color on the left panels represents mutants of Glut4/Dsred, the green color in the middle panels represents wild type Glut4/eGFP, and the yellow color in the right panels represents the colocalization of Glut4/eGFP and mutated Glut4/Dsred. The images were taken approximately through the middle of the cells and are representative of 4–5 independent experiments. The scale bar represents 10 μm.
Mentions: We previously noted the presence of sequence similarity between the extreme cytoplasmic C- terminal tail of Glut4 and a region within the amino terminus of the insulin responsive amino peptidase (IRAP), two membrane proteins that appear to share a major portion of their intracellular trafficking pathways in adipocytes (see Figure 1a). Two different sets of mutations within the IRM sequence motif (LXXLXPDEXD) were reported to result in aberrant subcellular targeting of Glut4 in the basal state and completely abolished its insulin-stimulated redistribution to the plasma membrane [34]. The possibility remained, however, that we had inadvertently introduced an unrelated and previously undefined dominant trafficking motif at the carboxy terminus of Glut4 in the two IRM mutants that were analyzed. We therefore examined the trafficking of 6 different single and double point mutations within the IRM sequence involving L500, L503, and P505 (see Figure 1b). The red fluorescent protein-tagged mutants were co-expressed in 3T3L1 adipocytes with green fluorescent protein-tagged wild type Glut4 in order to directly assess co-localization of the two different molecules within individual cells. We have previously demonstrated that wild type Glut4 targets indistinguishably whether it is tagged at its carboxyl-terminus with GFP or RFP, and that the tagged fusion proteins target very similarly to endogenous IRAP in 3T3L1 adipocytes [34]. Figure 2 shows that all 5 of the mutants in which either L500 and/or L503 were changed to alanine residues displayed aberrant targeting in the basal state compared to the wild type control, whereas the P505 mutant exhibited targeting that was indistinguishable from the wild type control under these conditions. Likewise, insulin-stimulated translocation of Glut4 to the cell periphery (representing presumptive plasma membrane insertion) was abolished in all 5 of the L500 and/or L503 mutants but remained intact in the single P505 point mutant (Figure 3). As observed previously, a much larger fraction of the aberrantly targeted mutants was present in large, dispersed, amorphous vesicles compared to the tagged wild-type Glut4, which was largely observed in small, diffuse, punctate structures and in a perinuclear compartment. Some co-localization between the mutants and wild-type transporters was observed in the perinuclear area.

Bottom Line: Insulin increases cellular glucose uptake and metabolism in the postprandial state by acutely stimulating the translocation of the Glut4 glucose transporter from intracellular membrane compartments to the cell surface in muscle and fat cells.The intracellular targeting of Glut4 is dictated by specific structural motifs within cytoplasmic domains of the transporter.We suggest that the IRM is critical for an early step in the sorting of Glut4 to insulin-responsive subcellular membrane compartments and that IRM mutants are miss-targeted to relatively large, amorphous membrane vesicles that may be involved in a degradation pathway for miss-targeted or miss-folded proteins or represent a transitional membrane compartment that Glut4 traverses en route to insulin responsive storage compartments.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology and Physiology, Washington University School of Medicine, St Louis, Missouri, United States of America.

ABSTRACT
Insulin increases cellular glucose uptake and metabolism in the postprandial state by acutely stimulating the translocation of the Glut4 glucose transporter from intracellular membrane compartments to the cell surface in muscle and fat cells. The intracellular targeting of Glut4 is dictated by specific structural motifs within cytoplasmic domains of the transporter. We demonstrate that two leucine residues at the extreme C-terminus of Glut4 are critical components of a motif (IRM, insulin responsive motif) involved in the sorting of the transporter to insulin responsive vesicles in 3T3L1 adipocytes. Light microscopy, immunogold electron microscopy, subcellular fractionation, and sedimentation analysis indicate that mutations in the IRM cause the aberrant targeting of Glut4 to large dispersed membrane vesicles that are not insulin responsive. Proteomic characterization of rapidly and slowly sedimenting membrane vesicles (RSVs and SSVs) that were highly enriched by immunoadsorption for either wild-type Glut4 or an IRM mutant revealed that the major vesicle fraction containing the mutant transporter (IRM-RSVs) possessed a relatively small and highly distinct protein population that was enriched for proteins associated with stress granules. We suggest that the IRM is critical for an early step in the sorting of Glut4 to insulin-responsive subcellular membrane compartments and that IRM mutants are miss-targeted to relatively large, amorphous membrane vesicles that may be involved in a degradation pathway for miss-targeted or miss-folded proteins or represent a transitional membrane compartment that Glut4 traverses en route to insulin responsive storage compartments.

Show MeSH
Related in: MedlinePlus