Identification and isolation of glycoproteins that translocate to the cell surface from GLUT4-enriched vesicles in an insulin-dependent fashion.

It has previously been shown that in fat cells, the intracellular reservoir of the glucose transporter isoform GLUT4 consists of a membrane vesicle population highly enriched in this transporter (approximately 15% of the protein content) that has a relatively simple protein pattern as revealed by SDS-polyacrylamide gel electrophoresis and silver staining (Zorzano, A., Wilkinson, W., Kotliar, N., Thoidis, G., Wadzinski, B. E., Ruoho, A. E., and Pilch, P. F. (1989) J. Biol. Chem. 264, 12358-12363). Upon exposure of adipocytes to insulin, the cell-surface (plasma membrane) content of GLUT4 is dramatically enhanced, and this transporter appears to continually cycle from intracellular vesicles to the plasma membrane. To identify other proteins that may recycle in a similar fashion to GLUT4 and that may participate in the insulin-dependent alteration in the cellular location of GLUT4 and other membrane proteins, we performed immunoadsorption of GLUT4-enriched vesicles together with biotinylation of membranes using a cell-impermeant analog of biotin. We find that immunoadsorbed GLUT4-containing vesicles can be fractionated into Triton X-100-soluble and -insoluble fractions. The first includes three major glycoprotein components with molecular masses of 110, 160, and 230 kDa and a few minor polypeptides with lower molecular masses. A Triton X-100-resistant fraction consists of GLUT4 and an approximately 25-kDa protein. All three major Triton-soluble proteins (110, 160, and 230 kDa) isolated from the immunoimmobilized vesicles on wheat germ agglutinin-agarose are strongly biotinylated in an insulin-dependent fashion, i.e. they cycle to and from the cell surface in an apparently identical manner to GLUT4. Sequence analysis of two tryptic fragments from p160 reveals that it is a novel protein containing sequence with no homology to known proteins.