Localization of the glucose phosphotransferase to a cytoplasmically accessible site on intracellular membranes.
UDP-glucose:glycoprotein glucose-1-phosphotransferase (Glc-phosphotransferase) catalyzes the transfer of alpha Glc-1-P from UDP-Glc to mannose residues on acceptor glycoproteins. The predominant acceptor for this transfer in rat liver is a glycoprotein of 62 kDa. This acceptor was labeled in liver homogenates through incubation with the 35S-labeled phosphorothioate analogue of UDP-Glc, and its distribution following differential centrifugation was compared to that of the glycoproteins labeled by CMP-[3H]N-acetylneuraminic acid. Whereas 94% of the 3H-labeled macromolecules fractionated to the microsomal pellet, 85% of the 35S-labeled 62-kDa glycoprotein was found in the high-speed supernatant. The distribution of the Glc-phosphotransferase was also examined following differential centrifugation, and the bulk of the activity was found in the 100,000 x g pellet. In contrast to results obtained with the lumenal microsomal markers 4 beta-galactosyltransferase and mannose-6-phosphatase, however, optimal activity of the Glc-phosphotransferase was not dependent on the disruption of microsomal vesicles by detergent. In addition, Glc-phosphotransferase was degraded by exogenous proteases in the absence of detergent, whereas the lumenal markers were not. We conclude, therefore, that the 62-kDa acceptor glycoprotein is cytoplasmic and is glycosylated by the Glc-phosphotransferase at a site accessible to the cytoplasm. This may prove to be a model for the topography of glycosylation of other cytoplasmic glycoproteins as well.
Srisomsap
C
C
Department of Cell Biology and Anatomy, University of Alabama, Birmingham 35294.
Richardson
K L
KL
Jay
J C
JC
Marchase
R B
RB
D000818:Animals; D002458:Cell Fractionation; D007425:Intracellular Membranes / Q000201:enzymology*; D007700:Kinetics; D008297:Male; D008862:Microsomes, Liver / Q000201:enzymology*; D008970:Molecular Weight; D010770:Phosphotransferases / Q000302:isolation & purification / Q000378:metabolism*; D051381:Rats; D017855:Transferases (Other Substituted Phosphate Groups)
D010770:Phosphotransferases; D017855:Transferases (Other Substituted Phosphate Groups); C037061:UDPglucose-glycoprotein glucose-1-phosphotransferase