Analysis of amino and carboxy terminal GLUT-4 targeting motifs in 3T3-L1 adipocytes using arm endosomal ablation technique

The targeting of the insulin-responsive glucose transporter, GLUT-4, to an intracellular compartment in adipocytes and muscle is one of the key featur es responsible for the unique insulin sensitivity of this transporter. Thro ugh expression of epitope-tagged GLUT-4 mutants in 3T3-L1 adipocytes, two m otifs have been identified as playing a central role in GLUT-4 targeting: F QQI in the amino terminus and a di-leucine motif in the carboxy terminus. T he goal of this study was to explore the role of these targeting motifs in the intracellular sorting of GLUT-4 using the Tf-HRP ablation technique. Th is technique provides a quantitative assessment of the amount of GLUT-LE lo cated in recycling endosomes. In basal adipocytes, we find that similar to 40% of GLUT-4 is ablated following Tf-HRP loading. In contrast, here we dem onstrate that the intracellular pool of a mutant in which F-5 was mutated t o A(5) is localized to the recycling endosomal pathway, suggesting that the amino terminal FQQI motif functions in trafficking GLUT-4 from early endos omes, In contrast, GLUT-4 in which (LL490)-L-489 was mutated to A(489)A(490 ) was localized predominantly to a nonablated compartment. These data imply a role for the di-leucine motif in sorting from a separate intracellular c ompartment, such as the TGN. Our findings are discussed within the context of a revised multicompartment model for GLUT-4 trafficking in adipocytes, i n which mutations in either the FQQI or LL motifs result in the altered sub cellular trafficking of GLUT-4 between multiple intracellular compartments.