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Subcellular Distribution of Low Molecular Weight Guanosine Triphosphate-Binding Proteins in Adipocytes: Colocalization with the Glucose Transporter Glut 4^*

Institut National de la Sante et de la Recherche Medicale U 145, Faculte de Medecine, Nice, France

Address requests for reprints to: Dr. Yannick Le Marchand-Brustel, INSERM U145, Faculte de Medecine, avenue de Valombrose, 06107 Nice Cedex 2, France.

Abstract

Insulin stimulation of glucose transport involves the translocation of vesicles containing the glucose transporter Glut 4 to the plasma membrane. Since low mol wt GTP-binding proteins (LMW-GTP-binding proteins) have been implicated in the regulation of vesicular trafficking, we have analyzed these proteins in adipocytes. Isolated adipocytes were incubated in the absence or presence of insulin before separation of plasma membranes (PM) and low density microsomes (LDM). [-³²P] GTP binding to proteins transferred to nitrocellulose after sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed specific and distinct subsets of proteins in the PM and LDM; those proteins were more abundant in PM than in LDM. [-³²P]GTP binding to these proteins was specific for the guanylnucleotides, since it was competed for by GTP and guanosine 5'-O-(3-thiotriphosphate), but not by ATP or adenosine 5'-O- (3-thiotriphosphate). The LMW-GTP-binding proteins were tightly associated with the membranes, as treatment with 1.5 M KC1 did not modify this association. The distribution of the LMW-GTP-binding proteins in the fractions and their affinity for guanylnucleotides were the same in control and insulintreated adipocytes. When the presence of G_i subunits was looked for with a specific antibody, G_i1 and G_i2 were found almost exclusively in PM. By contrast, the same antibody revealed the presence of a 100 kDa band in the LDM. Insulin treatment of adipocytes did not modify the amounts of those Gproteins in PM or LDM fractions, although it promoted the translocation of Glut 4 proteins from LDM to PM.

LDM fractions contain a specific subset of vesicles markedly enriched in Glut 4 molecules. When those vesicles were isolated from the total LDM fraction by immunoadsorption on highly specific antibodies to Glut 4 protein, LMW-GTP-binding proteins were found in the immune pellet. Those proteins were absent when immunoprecipitation was performed after solubilization of the vesicles with 1% Triton X-100. Our results strongly suggest that the vesicles containing the Glut 4 protein also contained LMW-GTP-binding proteins and indicate that these GTP-binding proteins could play a role in the exocytosis of the Glut 4-containing vesicles. (Endocrinology 129: 3343–3350, 1991)

Footnotes

^* This work was supported by grants from the French Association for the study of Myopathy, the Institut National de la Sante et de la Recherche Medicale, the University of Nice, the Fondation pour la Recherche Medicale, and the Region Provence Cote d'Azur.

Received May 7, 1991.

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