| Abstract
| - The targeting of the insulin-responsive glucose transporter, GLUT-4, to an intracellularcompartment in adipocytes and muscle is one of the key features responsible for the unique insulinsensitivity of this transporter. Through expression of epitope-tagged GLUT-4 mutants in 3T3-L1 adipocytes,two motifs have been identified as playing a central role in GLUT-4 targeting: FQQI in the amino terminusand a di-leucine motif in the carboxy terminus. The goal of this study was to explore the role of thesetargeting motifs in the intracellular sorting of GLUT-4 using the Tf−HRP ablation technique. Thistechnique provides a quantitative assessment of the amount of GLUT-4 located in recycling endosomes.In basal adipocytes, we find that ∼40% of GLUT-4 is ablated following Tf−HRP loading. In contrast,here we demonstrate that the intracellular pool of a mutant in which F5 was mutated to A5 is localized tothe recycling endosomal pathway, suggesting that the amino terminal FQQI motif functions in traffickingGLUT-4 from early endosomes. In contrast, GLUT-4 in which L489L490 was mutated to A489A490 waslocalized predominantly to a nonablated compartment. These data imply a role for the di-leucine motif insorting from a separate intracellular compartment, such as the TGN. Our findings are discussed withinthe context of a revised multicompartment model for GLUT-4 trafficking in adipocytes, in which mutationsin either the FQQI or LL motifs result in the altered subcellular trafficking of GLUT-4 between multipleintracellular compartments.
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