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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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Diagram of the overlapping protein compositions of the Glut4 and mutant IRM enriched vesicle fractions according to nano-LC-MS analysis.Each number in the circle represents the unique proteins in that fraction relative to the other. The underlined numbers in overlapping areas between two circles represent the number of shared proteins between the two fractions. The gray circle represents Glut4-RSV, the blank circle represents Glut4-SSV, the small gray shaded circle represents IRM-RSV, and the gray dotted circle represents IRM-SSV.
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pone-0068516-g007: Diagram of the overlapping protein compositions of the Glut4 and mutant IRM enriched vesicle fractions according to nano-LC-MS analysis.Each number in the circle represents the unique proteins in that fraction relative to the other. The underlined numbers in overlapping areas between two circles represent the number of shared proteins between the two fractions. The gray circle represents Glut4-RSV, the blank circle represents Glut4-SSV, the small gray shaded circle represents IRM-RSV, and the gray dotted circle represents IRM-SSV.

Mentions: Of the four vesicle fractions analyzed in the present study, only the Glut4 SSV fraction (traditionally referred to as the Glut4 low density microsomal (LDM) fraction) has been previously characterized by proteomic and immunological analyses. Our results confirm the presence of many proteins that have been reported to be present in the Glut4-SSV fraction by numerous studies [23], [44], [45] (proteins present in the Glut4-SSV fraction that have not been reported in previous studies are marked with an asterisk in Table S1). Among the best characterized of the known Glut4 vesicle proteins include sortilin, Glut1 (Slc2a1), mannose-6-P receptor, insulin- responsive aminopeptidase (Lnpep), transferrin receptor, IgF-II receptor, AS160 (TBC1D4), Vps45, carboxypeptidase D, Rab10, syntaxin16, syntaxin6, clathrin, and caveolin. Not surprisingly, most of these proteins are also present in the Glut4-RSV fraction (Table S2). The total RSV (“HDM”) fraction is enriched in markers for early biosynthetic compartments and probably contains compartments involved in the subcellular sorting of proteins associated with Glut4 intracellular vesicles. Of the 75 different proteins enriched at least 2-fold in either the Glut4-SSV or Glut4-RSV fractions, 33 (44%) of these were shared by the two compartments (see Figure 7).


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)

Diagram of the overlapping protein compositions of the Glut4 and mutant IRM enriched vesicle fractions according to nano-LC-MS analysis.Each number in the circle represents the unique proteins in that fraction relative to the other. The underlined numbers in overlapping areas between two circles represent the number of shared proteins between the two fractions. The gray circle represents Glut4-RSV, the blank circle represents Glut4-SSV, the small gray shaded circle represents IRM-RSV, and the gray dotted circle represents IRM-SSV.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3713040&req=5

pone-0068516-g007: Diagram of the overlapping protein compositions of the Glut4 and mutant IRM enriched vesicle fractions according to nano-LC-MS analysis.Each number in the circle represents the unique proteins in that fraction relative to the other. The underlined numbers in overlapping areas between two circles represent the number of shared proteins between the two fractions. The gray circle represents Glut4-RSV, the blank circle represents Glut4-SSV, the small gray shaded circle represents IRM-RSV, and the gray dotted circle represents IRM-SSV.
Mentions: Of the four vesicle fractions analyzed in the present study, only the Glut4 SSV fraction (traditionally referred to as the Glut4 low density microsomal (LDM) fraction) has been previously characterized by proteomic and immunological analyses. Our results confirm the presence of many proteins that have been reported to be present in the Glut4-SSV fraction by numerous studies [23], [44], [45] (proteins present in the Glut4-SSV fraction that have not been reported in previous studies are marked with an asterisk in Table S1). Among the best characterized of the known Glut4 vesicle proteins include sortilin, Glut1 (Slc2a1), mannose-6-P receptor, insulin- responsive aminopeptidase (Lnpep), transferrin receptor, IgF-II receptor, AS160 (TBC1D4), Vps45, carboxypeptidase D, Rab10, syntaxin16, syntaxin6, clathrin, and caveolin. Not surprisingly, most of these proteins are also present in the Glut4-RSV fraction (Table S2). The total RSV (“HDM”) fraction is enriched in markers for early biosynthetic compartments and probably contains compartments involved in the subcellular sorting of proteins associated with Glut4 intracellular vesicles. Of the 75 different proteins enriched at least 2-fold in either the Glut4-SSV or Glut4-RSV fractions, 33 (44%) of these were shared by the two compartments (see Figure 7).

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