Abstract
Insulin action on fat cell/skeletal muscle glucose transporter (GLUT4) redistribution to cell surface membranes appears to involve small GTP-binding proteins. It has been recently recognized that at least two GDP dissociation inhibitor (GDI) isoforms, GDI-1 and GDI-2, can bind and release GDP-bound Rab proteins from membranes (Shisheva, A., Südhof, T.C., and Czech, M. P. (1994) Mol. Cell. Biol. 14, 3459-3468). The present studies show that a strikingly higher level of GDI-2 fractionates with total membranes of COS-1 cells, Chinese hamster ovary cells, and 3T3-L1 adipocytes compared to GDI-1, which is virtually totally cytosolic. In 3T3-L1 adipocytes, most of the membrane-bound GDI-2 was present in a low density, intracellular membrane fraction. Immunodepletion of GLUT4-enriched vesicles from this membrane fraction also depleted significant amounts of GDI-2 proteins. Localization of both GDI-2 and GLUT4 in the same perinuclear regions of these cells was established by immunofluorescence microscopy, whereas GDI-1 displayed a diffuse, cytoplasmic distribution. Insulin acutely decreased both GLUT4 and GDI-2 protein levels in the low density microsomes by about 50%. Concomitantly, GLUT4 but not GDI-2 protein content of plasma membranes increased, suggesting release of GDI-2 into the cytoplasm in response to insulin. Taken together, these data suggest functional differences for the GDI-1 and GDI-2 protein isoforms, as well as a potential role of GDI-2 in the action of insulin on membrane movements.
Highlights
Differential Intracellular Localizations of GDP transporter GLUT4 [3,4,5,6,7,8]
Whereas both GLUTl and GLUT4 translocate to the adipocyte cell surface in response to insulin, thebulk of insulin-stimulated hexose flux is mediated by Dissociation Inhibitor Isoforms
Fig. 1shows the relative amountsof immunoreactive GDP dissociation inhibitor (GDI)-1 cytosol were obtained by sequential centrifugations: 5 min at 800 x g and GDI-2 proteins in thecytosolic uersus total membrane fracand 15min a t 200,000 x g in a BeckmanTL-100 Ultracentrifuge
Summary
Cell Cultures,Antibodies, and Fusion Proteins-3TB-Ll mouse fibroblasts, grown to confluence (100-mmplates or 18-mm round glass coverslips for immunofluorescence studies)in DMEM, containing 10%calf serum, 50 unitdml penicillin, and 50 pglml streptomycin sulfate, were differentiatedinto adipocytes as described previously [28].COS-1 and CHO-K1 cells were grown to confluence (100-mm plate) in DMEM or Ham’s F-12 medium, respectively, containing 10% FBS and the above antibiotics. Affinity-purified anti-GDI-1 andanti-GDI-2 antibodies were used in immunofluorescence microscopy studies. Cytosol (20 pg; C )and total membranes (3p0g;M ) , fractionated from the indicated cells, were resolved by SDS-PAGE (duplicate gels). GST-GDI-1 and GST-GDI-2 fusion proteinswere produced as de- Zmmunoblotting-For Western blot analysis, proteins (20-100 pg) scribed previously [21]. Their purity and concentrations were estimatesodlubilized in Lammli samplebuffer [32] were separatedon 10.5%SDS-. Cells were incubated with in our laboratory for more rapid fractionation of smaller amounts of affinity-purified anti-GDI-1or anti-GDI-2 antibodies GDI-1, GDI-2, or GLUT4 contents by Western blotting (triplicate gels)
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