Phosphoglucomutase in Saccharomyces cerevisiae is a cytoplasmic glycoprotein and the acceptor for a Glc-phosphotransferase.
UDP-glucose:glycoprotein glucose-1-phosphotransferase (Glc-phosphotransferase) catalyzes the transfer of Glc-1-P from UDP-Glc to mannose residues on acceptor glycoproteins. The predominant acceptor in vertebrates and Paramecium tetraurelia is a cytoplasmic 62-kDa glycoprotein. To determine if the yeast Saccharomyces cerevisiae also possesses Glc-phosphotransferase activity, a crude cellular lysate was incubated with [beta-32P]UDP-Glc and analyzed. A phosphoglycoprotein having an apparent molecular mass of 62 kDa (pgp62) was found to be the predominant labeled macromolecule. Reconstitution experiments determined that both a soluble and membrane fraction were required for labeling, and suggested that the Glc-phosphotransferase is membrane-associated while pgp62 is cytoplasmic. The reaction is evolutionarily conserved to the extent that rat liver Glc-phosphotransferase was capable of recognizing the yeast acceptor and vice versa. The yeast 62-kDa acceptor was purified, and partial amino acid sequences showed a high level of identity with rabbit muscle phosphoglucomutase. Subsequently, both yeast and rabbit muscle phosphoglucomutase were found to be acceptors in the Glc-phosphotransferase reaction. The label was found on a tryptic peptide distinct from that containing the enzyme's active site serine. When phosphoglucomutase was overexpressed, an increase was seen in Glc-phosphotransferase acceptor activity and in specific metabolic labeling of the acceptor by glucose and mannose.
Marchase
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Department of Cell Biology, University of Albama, Birmingham 35294.
Bounelis
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P
Brumley
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LM
Dey
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Browne
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Auger
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D
Fritz
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Kulesza
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Bedwell
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DM
D000595:Amino Acid Sequence; D000818:Animals; D003593:Cytoplasm / Q000201:enzymology; D005800:Genes, Fungal; D005947:Glucose / Q000378:metabolism; D006023:Glycoproteins / Q000378:metabolism*; D008358:Mannose / Q000378:metabolism; D008715:Methionine / Q000378:metabolism; D008969:Molecular Sequence Data; D009132:Muscles / Q000201:enzymology; D009154:Mutation; D010733:Phosphoglucomutase / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D010761:Phosphorus Radioisotopes; D010770:Phosphotransferases / Q000378:metabolism*; D010957:Plasmids; D011817:Rabbits; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D017855:Transferases (Other Substituted Phosphate Groups); D014532:Uridine Diphosphate Glucose / Q000378:metabolism
D006023:Glycoproteins; D010761:Phosphorus Radioisotopes; D008715:Methionine; D010770:Phosphotransferases; D017855:Transferases (Other Substituted Phosphate Groups); C037061:UDPglucose-glycoprotein glucose-1-phosphotransferase; D010733:Phosphoglucomutase; D005947:Glucose; D008358:Mannose; D014532:Uridine Diphosphate Glucose