Evidence for interaction of yeast protein kinase C with several subunits of oligosaccharyl transferase.
Evidence for interaction of yeast protein kinase C with several subunits of oligosaccharyl transferase.
复制标题
酵母蛋白激酶 C 与寡糖基转移酶的几个亚基相互作用的证据。
DOI：
10.1093/glycob/10.7.737
复制
发表时间：
2000
期刊：
影响因子：
4.3
通讯作者：
Lennarz,WJ
中科院分区：
文献类型：
--
作者：
Park,H;Lennarz,WJ
酵母寡糖基转移酶（Oligosaccharyltransferase，OT）是由位于内质网（Endoplasmic reticulum，ER）的8个跨膜蛋白组成的酶复合物。利用双杂交文库筛选OT中潜在的蛋白质-蛋白质相互作用的研究表明，蛋白激酶C（Pkc 1 p）与几个OT亚基的内腔结构域相互作用。额外的遗传实验表明，两个OT亚基的过表达挽救了由Pkc 1活性位点突变体的过表达引起的生长缺陷，这意味着PKC 1和OT之间存在特定的遗传相互作用。这些体内发现得到了体外研究的补充，体外研究表明，几个OT亚基与谷胱甘肽S-转移酶通过其C-末端连接到Pkc 1 p的融合蛋白结合。OT活性的测定，其中一个简单的受体肽的糖基化进行了测定，在微粒体从野生型和pkc 1空揭示了一个50%的活性减少在微粒体从空菌株。相比之下，含有已知在PKC 1-MAP激酶途径中Pkc 1 p下游的另外两个基因的无效突变的菌株具有与野生型细胞相当的OT活性水平。这些在体内和体外实验表明，在酵母细胞Pkc 1 p可能参与调节蛋白质的N-糖基化。
Oligosaccharyltransferase (OT) inSaccharomyces cerevisiaeis an enzyme complex consisting of 8 transmembrane proteins located in the endoplasmic reticulum (ER). Studies on potential protein–protein interactions in OT using a two-hybrid library screen revealed that protein kinase C (Pkc1p) interacted with the lumenal domains of several OT subunits. Additional genetic experiments revealed that overexpression of two OT subunits rescued the growth defect caused by overexpression of a Pkc1 active site mutant, implying that there are specific genetic interactions between PKC1 and OT. Thesein vivofindings were complemented byin vitrostudies that showed that several of the OT subunits bound to a fusion protein consisting of glutathione S-transferase linked via its C-terminus to Pkc1p. Assays of OT activity, in which glycosylation of a simple acceptor peptide was assayed in microsomes from wild-type and a pkc1 null revealed a 50% reduction in activity in the microsomes from the null strain. In contrast, strains containing null mutations of two other genes known to be downstream of Pkc1p in the PKC1-MAP kinase pathway had a level of OT activity comparable to that of wild-type cells. Thesein vivoandin vitroexperiments suggest that in yeast cells Pkc1p may be involved in regulation of the N-glycosylation of proteins.