Hong G, Trumbly R J, Reimann E M, Schlender K K
Department of Biochemistry and Molecular Biology, Department of Pharmacology, Medical College of Ohio, 3035 Arlington Avenue, Toledo, Ohio 43614-5804, USA.
Arch Biochem Biophys. 2000 Apr 15;376(2):288-98. doi: 10.1006/abbi.2000.1715.
Protein phosphatase 1 (PP1) is one of the major protein phosphatases in eukaryotic cells. PP1 activity is believed to be controlled by the interaction of PP1 catalytic subunit with various regulatory subunits. The essential gene GLC7 encodes the PP1 catalytic subunit in Saccharomyces cerevisiae. In this study, full-length GLC7(1-312), C-terminal deletion mutants, and C-terminally poly-his tagged mutants were constructed and expressed in a GLC7 knockout strain of S. cerevisiae. Viability studies of the GLC7 knockout strains carrying the plasmids expressing GLC7 C-terminal deletion mutants and their tagged forms showed that the mutants 1-295 and 1-304 were functional, whereas the mutant 1-245 was not. The C-terminally poly-his tagged Glc7p with and without an N-terminal hemagglutinin (HA) tag was partially purified by immobilized Ni(2+) affinity chromatography and further analyzed by gel filtration and ion exchange chromatography. Phosphatase activity assays, SDS-PAGE, and Western blot analyses of the chromatographic fractions suggested that the Glc7p associated with regulatory subunits in vivo. A 40-kDa protein was copurified with tagged Glc7p through several chromatographic procedures. Monoclonal antibody against the HA tag coimmunoprecipitated the tagged Glc7p and the 40-kDa protein. This protein was further purified by a reverse phase HPLC column. Analysis by CNBr digestion, peptide sequencing, and electrospray mass spectrometry showed that this 40-kDa protein is Sds22p, one of the proteins proposed to be a regulatory subunit of Glc7. These results demonstrate that Sds22p forms a complex with Glc7p and that Sds22p:Glc7p is a stable isolatable form of yeast PP1.
蛋白磷酸酶1（PP1）是真核细胞中主要的蛋白磷酸酶之一。人们认为PP1的活性受PP1催化亚基与各种调节亚基相互作用的控制。必需基因GLC7编码酿酒酵母中的PP1催化亚基。在本研究中，构建了全长GLC7(1 - 312)、C端缺失突变体以及C端多聚组氨酸标签突变体，并在酿酒酵母的GLC7基因敲除菌株中进行表达。对携带表达GLC7 C端缺失突变体及其标签形式质粒的GLC7基因敲除菌株进行的活力研究表明，突变体1 - 295和1 - 304具有功能，而突变体1 - 245没有功能。通过固定化Ni(2+)亲和层析对带有和不带有N端血凝素（HA）标签的C端多聚组氨酸标记的Glc7p进行了部分纯化，并通过凝胶过滤和离子交换层析进一步分析。对层析级分进行的磷酸酶活性测定、SDS - PAGE和蛋白质印迹分析表明，Glc7p在体内与调节亚基相关联。通过几种层析方法，一种40 kDa的蛋白质与标记的Glc7p共纯化。抗HA标签的单克隆抗体共免疫沉淀了标记的Glc7p和40 kDa的蛋白质。该蛋白质通过反相HPLC柱进一步纯化。通过CNBr消化、肽测序和电喷雾质谱分析表明，这种40 kDa的蛋白质是Sds22p，它是被认为是Glc7调节亚基的蛋白质之一。这些结果表明Sds22p与Glc7p形成复合物，并且Sds22p:Glc7p是酵母PP1的一种稳定可分离形式。
