Isolation of the GSY1 gene encoding yeast glycogen synthase and evidence for the existence of a second gene.
Glycogen synthase preparations from Saccharomyces cerevisiae contained two polypeptides of molecular weights 85,000 and 77,000. Oligonucleotides based on protein sequence were utilized to clone a S. cerevisiae glycogen synthase gene, GSY1. The gene would encode a protein of 707 residues, molecular mass 80,501 daltons, with 50% overall identity to mammalian muscle glycogen synthases. The amino-terminal sequence obtained from the 85,000-dalton species matched the NH2 terminus predicted by the GSY1 sequence. Disruption of the GSY1 gene resulted in a viable haploid with glycogen synthase activity, and purification of glycogen synthase from this mutant strain resulted in an enzyme that contained the 77,000-dalton polypeptide. Southern hybridization of genomic DNA using the GSY1 coding sequence as a probe revealed a second weakly hybridizing fragment, present also in the strain with the GSY1 gene disrupted. However, the sequences of several tryptic peptides derived from the 77,000-dalton polypeptide were identical or similar to the sequence predicted by the GSY1 gene. The data are explained if S. cerevisiae has two glycogen synthase genes encoding proteins with significant sequence similarity The protein sequence predicted by the GSY1 gene lacks the extreme NH2-terminal phosphorylation sites of the mammalian enzymes. The COOH-terminal phosphorylated region of the mammalian enzyme over-all displayed low identity to the yeast COOH terminus, but there was homology in the region of the mammalian phosphorylation sites 3 and 4. Three potential cyclic AMP-dependent protein kinase sites are located in this region of the yeast enzyme. The region of glycogen synthase likely to be involved in covalent regulation are thus more variable than the catalytic center of the molecule.
Farkas
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Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, Indianapolis 46223.
Hardy
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DePaoli-Roach
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Roach
P J
PJ
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D005800:Genes, Fungal; D006006:Glycogen Synthase / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D007527:Isoenzymes / Q000235:genetics* / Q000302:isolation & purification; D008099:Liver / Q000201:enzymology; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009132:Muscles / Q000201:enzymology; D015345:Oligonucleotide Probes; D011817:Rabbits; D051381:Rats; D015183:Restriction Mapping; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics*; D012689:Sequence Homology, Nucleic Acid
D007527:Isoenzymes; D015345:Oligonucleotide Probes; D006006:Glycogen Synthase