Self-assembly of Glut4 Storage Vesicles during Differentiation of 3T3-L1 Adipocytes

Abstract

Glut4 storage vesicles (GSVs) represent translocationcompetent 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.Insulin increases glucose uptake in fat and skeletal muscle tissues by translocating intracellular Glut4-containing vesicles (also called IRVs for insulin-responsive vesicles and GSVs 4 for Glut4 storage vesicles) to the plasma membrane (1, 2). One of the major protein components of the GSVs is insulin-responsive aminopeptidase, or IRAP (3-5).Although the biological role of IRAP is not yet clear (6), recent evidence indicates that the presence of IRAP in the GSVs confers insulin responsiveness to this compartment (7). Thus, IRAP has been shown to interact with AS160, a Rab GTPaseactivating protein that is phosphorylated by Akt and may couple Glut4 traffic to the insulin signaling pathway (8,9). IRAP also interacts with other proteins that have been implemented in regulation of Glut4 translocation, such as p115 (10) and tankyrase (7). Interestingly, microinjection into 3T3-L1 adipocytes of the recombinant peptide corresponding to the cytoplasmic tail of IRAP causes translocation of Glut4, presumably by competition with putative GSV anchors (11).In addition to Glut4 and IRAP, GSVs incorporate a putative sorting receptor, sortilin (12-14), VAMP2 (15), several recycling receptors, and other membrane proteins that may or may not represent essential vesicular components (8). In any case, the presence of Glut4, IRAP, and sortilin as the most prominent components of the GSV membrane is a unique feature of this compartment that differs from other vesicular carriers in the cell. A challenging problem is to determine how these structurally and functionally different proteins become co-localized in the cell and form a unique, highly specialized vesicular carrier. To this end, we...

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