CHARACTERIZATION OF A MUTANT GLUT4 LACKING THE N-GLYCOSYLATION SITE -STUDIES IN TRANSFECTED RAT ADIPOSE-CELLS

GLUT4, the insulin-responsive glucose transporter expressed primarily in muscle and adipose tissue, contains a single N-glycosylation sire. We characterized a mutant GLUT4 lacking the N-glycosylation site (Asn( 57) --> Gin) in primary cultures of rat adipose cells, We transiently transfected cells with expression vectors for epitope-tagged GLUT4 con taining either wild-type (GLUT4-HA) or mutant (GLN57-HA) cDNA sequence s, Expression of GLN57-HA in adipose cells was similar to 10-fold lowe r than for GLUT4-HA even though mRNA levels for bath recombinant trans porters were comparable, Biosynthetic labeling studies showed markedly decreased incorporation of [S-35]-methionine/cysteine into GLN57-HA r elative to GLUT4-HA consistent with either a decreased synthetic rate or accelerated degradation of GLN57-HA. Interestingly, transient trans fection of GLUT4-HA and GLN57-HA in COS-7 cells (which do not express endogenous GLUT4) resulted in comparable levels of protein expression for both transporters. Thus, in the physiologically relevant adipose c ell, glycosylation of GLUT4 appears to play an important functional ro le. (C) 1996 Academic Press, Inc.