Analysis of Amino and Carboxy Terminal GLUT-4 Targeting Motifs in 3T3-L1 Adipocytes Using an Endosomal Ablation Technique

Abstract

The targeting of the insulin-responsive glucose transporter, GLUT-4, to an intracellular compartment in adipocytes and muscle is one of the key features responsible for the unique insulin sensitivity 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 terminus and a di-leucine motif in the carboxy terminus. The 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. This technique 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 to the recycling endosomal pathway, suggesting that the amino terminal FQQI motif functions in trafficking GLUT-4 from early endosomes. In contrast, GLUT-4 in which L⁴⁸⁹L⁴⁹⁰ was mutated to A⁴⁸⁹A⁴⁹⁰ was localized predominantly to a nonablated compartment. These data imply a role for the di-leucine motif in sorting from a separate intracellular compartment, such as the TGN. Our findings are discussed within the context of a revised multicompartment model for GLUT-4 trafficking in adipocytes, in which mutations in either the FQQI or LL motifs result in the altered subcellular trafficking of GLUT-4 between multiple intracellular compartments.