Lin J, Addison R
Department of Biochemistry, University of Tennessee, Memphis 38163.
J Biol Chem. 1994 Feb 4;269(5):3887-90.
The Neurospora plasma membrane H(+)-ATPase is a polytopic integral membrane protein. To localize transmembrane segments, mutants were constructed that contained the amino and carboxyl termini of the H(+)-ATPase with putative transmembrane segment. A stretch of amino acid residues from yeast invertase that has three consensus N-linked glycosylation sites was placed carboxyl terminal of the putative transmembrane segment. RNA transcripts of these mutants were translated in a Neurospora in vitro system that was supplemented with microsomes from Neurospora. By the criteria of glycosylation of the polypeptide chain, resistance to extraction at pH 11.5, and protection from proteinase K digestion, only one transmembrane segment could be identified within the amino acid residues 272-314 of the primary sequence of the H(+)-ATPase.
粗糙脉孢菌质膜H(+)-ATP酶是一种多次跨膜整合膜蛋白。为了定位跨膜区段，构建了一些突变体，这些突变体包含H(+)-ATP酶的氨基末端和羧基末端以及假定的跨膜区段。一段来自酵母蔗糖酶的具有三个共有N-连接糖基化位点的氨基酸残基被置于假定跨膜区段的羧基末端。这些突变体的RNA转录本在添加了粗糙脉孢菌微粒体的粗糙脉孢菌体外系统中进行翻译。根据多肽链糖基化、在pH 11.5下的提取抗性以及对蛋白酶K消化的抗性标准，在H(+)-ATP酶一级序列的氨基酸残基272-314内只能鉴定出一个跨膜区段。
