SUBCELLULAR TRAFFICKING KINETICS OF GLUT4 MUTATED AT THE N-TERMINUS AND C-TERMINUS

The glucose transporter isoform, GLUT4, has been expressed in Chinese hamster ovary clones and its subcellular trafficking has been determin ed following labelling at the cell surface with the impermeant bis-man nose photolabel, yl)benzoyl-1,3-bis(D-mannos-4-yloxy)-2-propylamine (A TB-BMPA). ATB-BMPA-tagged GLUT4 leaves the cell surface rapidly and eq uilibrates to give an internal/surface distribution ratio of approx, 3 .5 after 60 min. GLUT4 in which the N-terminal phenylalanine-5 and glu tamine-6 are mutated to alanine N-(FQ-AA) and in which the C-terminal leucine-489 and -490 are mutated to alanine C-(LL-AA) have low interna l/surface ratios of 0.64 and 1.24 respectively. If all cell-surface tr ansporters are able to recycle, as would be the case for a two-pool re cycling model with a single intracellular pool, then analysis suggests that the wild-type GLUT4 distribution ratio is dependent on endocytos is and exocytosis rate constants of 0.074 and 0.023 min(-1) These valu es are similar, but not identical, to those found for GLUT4 traffickin g in adipocytes. The distribution of the N-(FQ-AA) transporter appears to be due to a decrease in endocytosis with reduced intracellular ret ention, while the distribution of the C-(LL-AA) transporter appears to be mainly due to poor intracellular retention. These results are also considered in terms of a consecutive intracellular pool model in whic h GLUT4 targeting domains alter the distribution between recycling end osomes and a slowly recycling compartment. In this case the more rapid apparent exocytosis of the mutated GLUT4s is due to their failure to reach a slowly recycling compartment with a consequent return to the p lasma membrane by default. It is suggested that overexpression of tran sporters increases the proportion that are recycled in this way, Wortm annin is shown to decrease glucose transport activity and cell-surface photolabelled transporters in a manner consistent with an inhibition of transporter recycling. Studies on the rate of loss of transport act ivity and ATB-BMPA-tagged transporter in wortmannin-treated cells conf irm that the N-(FQ-AA) mutant is endocytosed more slowly than the wild type GLUT4. Taken together, these results suggest that mutation at eit her the N- or the C-terminal domain can reduce movement to a slowly re cycling intracellular compartment but that neither domain alone is ent irely sufficient to produce wild-type GLUT4 trafficking behaviour.