Self-assembly of Glut4 storage vesicles during differentiation of 3T3-L1 adipocytes.
Glut4 storage vesicles (GSVs) represent translocation-competent vesicular carriers in fat and skeletal muscle cells that deliver Glut4 to the plasma membrane in response to insulin stimulation. GSVs include three major cargo proteins: Glut4, insulin-responsive aminopeptidase (IRAP), and sortilin. Previous work has suggested that the lumenal interaction between Glut4 and sortilin and the cytoplasmic interaction between sortilin and GGA adaptors play an important role in recruitment of Glut4 into the GSVs. However, the mechanism of IRAP targeting to this compartment remains unknown. To address this question, we show that in differentiating adipocytes IRAP enters the GSVs from the "donor" membranes on day 3 of differentiation. Forced expression of sortilin in undifferentiated cells does not recruit IRAP into the vesicles. However, double expression of sortilin and Glut4 reconstitutes functional GSVs that incorporate endogenous IRAP. To explain this process, we show by a yeast two-hybrid system and chemical cross-linking that the lumenal domain of IRAP can interact with the lumenal loop of Glut4. IRAP without the lumenal domain is faithfully targeted to the donor membranes but has significantly lower insulin responsiveness than full-length IRAP. We suggest that lumenal interactions between Glut4 and IRAP play an important role in the assembly of the GSVs.
Shi
Jun
J
Boston University School of Medicine, Boston, Massachusetts 02118, USA.
Huang
Guanrong
G
Kandror
Konstantin V
KV
D041721:3T3-L1 Cells; D033942:Adaptor Proteins, Vesicular Transport; D017667:Adipocytes / Q000378:metabolism*; D000818:Animals; D002454:Cell Differentiation; D003432:Cross-Linking Reagents / Q000494:pharmacology; D010122:Cystinyl Aminopeptidase / Q000378:metabolism*; D003600:Cytosol / Q000378:metabolism; D051275:Glucose Transporter Type 4 / Q000378:metabolism / Q000502:physiology*; D007328:Insulin / Q000378:metabolism; D008562:Membrane Glycoproteins / Q000378:metabolism; D051379:Mice; D008954:Models, Biological; D009419:Nerve Tissue Proteins / Q000378:metabolism; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D020798:Two-Hybrid System Techniques; D000099183:Sortilin
D033942:Adaptor Proteins, Vesicular Transport; D003432:Cross-Linking Reagents; D010122:Cystinyl Aminopeptidase; D051275:Glucose Transporter Type 4; D007328:Insulin; D008562:Membrane Glycoproteins; D009419:Nerve Tissue Proteins; D000099183:Sortilin; C495693:Slc2a4 protein, mouse; C105964:leucyl-cystinyl aminopeptidase