Subcellular trafficking kinetics of GLU4 mutated at the N- and C-terminal.
The glucose transporter isoform, GLUT4, has been expressed in Chinese hamster clones and its subcellular trafficking has been determined following labelling at the cell surface with the impermeant bis-mannose photolabel, 2-N-(1-azi-2,2,2-trifluoroethyl)benzoyl-1,3-bis(D-mannos -4-yloxy)-2-propylamine (ATM-BMPA). ATM-BMPA-tagged GLUT4 leaves the cell surface rapidly and equilibrates to give an internal/surface distribution ratio of approx. 3.5 after 60 min. GLUT4 in which the N-terminal phenylalanine-5 and glutamine-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 internal/surface ratios of 0.64 and 1.24 respectively. If all cell-surface transporters are able to recycle, as would be the case for a two-pool recycling model with a single intracellular pool, then analysis suggests that the wild-type GLUT4 distribution ratio is dependent on endocytosis and exocytosis rate constants of 0.074 and 0.023 min(-1). These values are similar, but not identical, to those found for GLUT4 trafficking in adipocytes. The distribution of the N-(FQ-AA) transporter appears to be due to a decrease in endocytosis with reduced intracellular retention, 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 which GLUT4 targeting domains alter the distribution between recycling endosomes and a slowly recycling compartment. In this case the more rapid apparent exocytosis of the mutated GLUT4 is due to their failure to reach a slowly recycling compartment with a consequent return to the plasma membrane by default. It is suggested that overexpression of transporters increases the proportion that are recycled in this way. Wortmannin 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 activity and ATB-BMPA-tagged transporter in wortmannin-treated cells confirm that the N-(FQ-AA) mutant is endocytosed more slowly than the wild-type GLUT4. Taken together, these results suggest that the mutation at either the N- or the C-terminal domain can reduce movement to a slowly recycling intracellular compartment but that neither domain alone is entirely sufficient to produce wild-type GLUT4 trafficking behaviour.
Araki
S
S
The Second Department of Internal Medicine, Kobe University School of Medicine, Japan.
Yang
J
J
Hashiramoto
M
M
Tamori
Y
Y
Kasuga
M
M
Holman
G D
GD
D000595:Amino Acid Sequence; D000730:Androstadienes / Q000494:pharmacology; D000818:Animals; D001483:Base Sequence; D016466:CHO Cells / Q000187:drug effects / Q000378:metabolism / Q000502:physiology; D006224:Cricetinae; D018076:DNA, Complementary / Q000235:genetics; D051272:Glucose Transporter Type 1; D051275:Glucose Transporter Type 4; D006801:Humans; D007328:Insulin / Q000494:pharmacology; D007329:Insulin Antagonists / Q000494:pharmacology; D007700:Kinetics; D008969:Molecular Sequence Data; D009004:Monosaccharide Transport Proteins / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D009124:Muscle Proteins; D009154:Mutation; D013347:Subcellular Fractions / Q000378:metabolism; D014162:Transfection; D000077191:Wortmannin
D000730:Androstadienes; D018076:DNA, Complementary; D051272:Glucose Transporter Type 1; D051275:Glucose Transporter Type 4; D007328:Insulin; D007329:Insulin Antagonists; D009004:Monosaccharide Transport Proteins; D009124:Muscle Proteins; C495677:SLC2A1 protein, human; C495692:SLC2A4 protein, human; D000077191:Wortmannin