Farkas I, Hardy T A, DePaoli-Roach A A, Roach P J
Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, Indianapolis 46223.
J Biol Chem. 1990 Dec 5;265(34):20879-86.
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.
来自酿酒酵母的糖原合酶制剂含有分子量分别为85,000和77,000的两种多肽。基于蛋白质序列的寡核苷酸被用于克隆酿酒酵母糖原合酶基因GSY1。该基因将编码一个由707个残基组成、分子量为80,501道尔顿的蛋白质，与哺乳动物肌肉糖原合酶的整体一致性为50%。从85,000道尔顿的蛋白中获得的氨基末端序列与GSY1序列预测的NH2末端相匹配。GSY1基因的破坏导致了一个具有糖原合酶活性的存活单倍体，从该突变菌株中纯化糖原合酶得到了一种含有77,000道尔顿多肽的酶。用GSY1编码序列作为探针进行基因组DNA的Southern杂交，发现了第二个弱杂交片段，在GSY1基因被破坏的菌株中也存在。然而，从77,000道尔顿多肽衍生的几个胰蛋白酶肽段的序列与GSY1基因预测的序列相同或相似。如果酿酒酵母有两个编码具有显著序列相似性的蛋白质的糖原合酶基因，那么这些数据就能得到解释。GSY1基因预测的蛋白质序列缺乏哺乳动物酶的极端NH2末端磷酸化位点。哺乳动物酶的COOH末端磷酸化区域与酵母COOH末端的整体一致性较低，但在哺乳动物磷酸化位点3和4的区域存在同源性。酵母酶的这一区域有三个潜在的环磷酸腺苷依赖性蛋白激酶位点。因此，糖原合酶中可能参与共价调节的区域比分子的催化中心更具变异性。
