Abstract
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.
Highlights
The presence of glucose transporter isoform 4 (Glut4), insulin-responsive aminopeptidase (IRAP), and sortilin as the most prominent components of the Glut4 storage vesicles (GSVs) membrane is a unique feature of this compartment that differs from other vesicular carriers in the cell
Upon ectopic expression in undifferentiated 3T3-L1 pre-adipocytes, Glut4 (Fig. 2A) is targeted to the same perinuclear compartment, suggesting that the later may serve as the donor compartment for the formation of the GSVs upon cell differentiation
In agreement with this hypothesis, biochemical fractionation of differentiated adipocytes shows that both IRAP (Fig. 3A) and ectopically expressed myc7-Glut4 [14] re-distribute from heavy donor membranes to much lighter GSVs
Summary
GSVs during Differentiation of 3T3-L1 Adipocytes expressed in undifferentiated 3T3-L1 cells is targeted to small vesicles but has only a minor effect on the recruitment of endogenous IRAP to this compartment. Wild type 3T3-L1 pre-adipocytes do not express sortilin or Glut4 and have very little IRAP in the GSV fraction (see Fig. 3B).
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