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J. Biol. Chem., Vol. 283, Issue 44, 30311-30321, October 31, 2008

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

From the Boston University School of Medicine, Boston, Massachusetts 02118

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.

Received for publication, July 8, 2008 , and in revised form, August 18, 2008.

^* This work was supported, in whole or in part, by National Institutes of Health Grant DK52057 and DK56736. This work was also supported by a Research Award from the American Diabetes Association (to K. V. K.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ Both authors contributed equally to the study.

² Current address: Novartis Institutes for Biomedical Research, 100 Technology Square, Cambridge, MA 02139.

³ To whom correspondence should be addressed: Dept. of Biochemistry, Boston University School of Medicine, K124D, 715 Albany St., Boston, MA 02118. Tel.: 617-638-5049; Fax: 617-638-5339; E-mail: kandror{at}biochem.bumc.bu.edu.

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