Localization of the glucose phosphotransferase to a cytoplasmically accessible site on intracellular membranes

• Published in: The Journal of biological chemistry Vol. 263; no. 33; pp. 17792 - 17797
• Main Authors: Srisomsap, C, Richardson, K L, Jay, J C, Marchase, R B
• Format: Journal Article
• Language: English
• Published: Bethesda, MD Elsevier Inc 25.11.1988; American Society for Biochemistry and Molecular Biology
• Subjects: Analytical, structural and metabolic biochemistry; Animals; Biological and medical sciences; Cell Fractionation; Fundamental and applied biological sciences. Psychology; Glycoproteins; Intracellular Membranes - enzymology; Kinetics; liver; Male; Microsomes, Liver - enzymology; Molecular Weight; Phosphotransferases - isolation & purification; Phosphotransferases - metabolism; Proteins; Rats; Transferases (Other Substituted Phosphate Groups); UDP-glucose:glycoprotein glucose-1-phosphotransferase; Vertebrata; Mammalia; Rat; Enzyme; Rodentia; Biosynthesis; Glycoproteins; Localization
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/S0021-9258(19)77905-8

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.