Lin J, Addison R
Department of Biochemistry, University of Tennessee, Memphis 38163.
J Biol Chem. 1995 Mar 24;270(12):6942-8. doi: 10.1074/jbc.270.12.6942.
To localize transmembrane segments in the carboxyl-terminal third of the Neurospora plasma membrane H(+)-ATPase, we constructed fusion proteins on the cDNA level. These contained DNA fragments encoding hydrophilic residues of the amino and carboxyl termini of the H(+)-ATPase with a DNA fragment encoding the putative transmembrane segment. To report translocation into microsomes, a DNA fragment encoding three consensus N-linked glycosylation sites was engineered carboxyl-terminal to the putative transmembrane segment. Fusion proteins were synthesized in a Neurospora in vitro translation system supplemented with homologous microsomes. By the criteria of glycosylation of fusion proteins by microsomes, sedimentation of products with microsomes after alkaline extraction, and analysis of protected fragments generated from proteinase K digestion of integrated products, we localized six transmembrane segments in the carboxyl-terminal third of the H(+)-ATPase. These results support a 10-segment model of the Neurospora H(+)-ATPase.
为了定位粗糙脉孢菌质膜H(+)-ATP酶羧基末端三分之一区域的跨膜片段，我们在cDNA水平构建了融合蛋白。这些融合蛋白包含编码H(+)-ATP酶氨基末端和羧基末端亲水性残基的DNA片段，以及编码推定跨膜片段的DNA片段。为了报告向微粒体的转运，在推定跨膜片段的羧基末端设计了一个编码三个共有N-连接糖基化位点的DNA片段。融合蛋白在补充了同源微粒体的粗糙脉孢菌体外翻译系统中合成。根据微粒体对融合蛋白的糖基化标准、碱性提取后产物与微粒体的沉降以及对整合产物经蛋白酶K消化产生的受保护片段的分析，我们在H(+)-ATP酶羧基末端三分之一区域定位了六个跨膜片段。这些结果支持粗糙脉孢菌H(+)-ATP酶的十片段模型。
