The C-terminus of GLUT4 targets the transporter to the perinuclear compartment but not to the insulin-responsive vesicles.

Postprandial blood glucose clearance is mediated by GLUT4 (glucose transporter 4) which is translocated from an intracellular storage pool to the plasma membrane in response to insulin. The nature of the intracellular storage pool of GLUT4 is not well understood. Immunofluorescence staining shows that, under basal conditions, the major population of GLUT4 resides in the perinuclear compartment. At the same time, biochemical fractionation reveals that GLUT4 is localized in IRVs (insulin-responsive vesicles). The relationship between the perinuclear GLUT4 compartment and the IRVs is not known. In the present study, we have exchanged the C-termini of GLUT4 and cellugyrin, another vesicular protein that is not localized in the IRVs and has no insulin response. We have found that GLUT4 with the cellugyrin C-terminus loses its specific perinuclear localization, whereas cellugyrin with the GLUT4 C-terminus acquires perinuclear localization and becomes co-localized with GLUT4. This, however, is not sufficient for the effective entry of the latter chimaera into the IRVs as only a small fraction of cellugyrin with the GLUT4 C-terminus is targeted to the IRVs and is translocated to the plasma membrane in response to insulin stimulation. We suggest that the perinuclear GLUT4 storage compartment comprises the IRVs and the donor membranes from which the IRVs originate. The C-terminus of GLUT4 is required for protein targeting to the perinuclear donor membranes, but not to the IRVs.

Golgi-localized, gamma-ear-containing, Arf-binding protein adaptors mediate insulin-responsive trafficking of glucose transporter 4 in 3T3-L1 adipocytes.

Small glucose transporter 4 (Glut4)-containing vesicles represent the major insulin-responsive compartment in fat and skeletal muscle cells. The molecular mechanism of their biogenesis is not yet elucidated. Here, we studied the role of the newly discovered family of monomeric adaptor proteins, GGA (Golgi-localized, gamma-ear-containing, Arf-binding proteins), in the formation of small Glut4 vesicles and acquisition of insulin responsiveness in 3T3-L1 adipocytes. In these cells, all three GGA isoforms are expressed throughout the differentiation process. In particular, GGA2 is primarily present in trans-Golgi network and endosomes where it demonstrates a significant colocalization with the recycling pool of Glut4. Using the techniques of immunoadsorption as well as glutathione-S-transferase pull-down assay we found that Glut4 vesicles (but not Glut4 per se) interact with GGA via the Vps-27, Hrs, and STAM (VHS) domain. Moreover, a dominant negative GGA mutant inhibits formation of Glut4 vesicles in vitro. To study a possible role of GGA in Glut4 traffic in the living cell, we stably expressed a dominant negative GGA mutant in 3T3-L1 adipocytes. Formation of small insulin-responsive Glut4-containing vesicles and insulin-stimulated glucose uptake in these cells were markedly impaired. Thus, GGA adaptors participate in the formation of the insulin-responsive vesicular compartment from the intracellular donor membranes both in vivo and in vitro.