Lin J, Addison R
Department of Biochemistry, University of Tennessee, Memphis 38163.
J Biol Chem. 1995 Mar 24;270(12):6935-41. doi: 10.1074/jbc.270.12.6935.
The Neurospora plasma membrane H(+)-ATPase belongs to a family of cation-motive porters called P-type ATPases. Putative transmembrane segments of these enzymes contain one or more charged residues. Conditions were determined by which a transmembrane segment with charged residues is integrated into its cognate membrane. We constructed fusion proteins flanked by the hydrophilic domains of the amino and carboxyl termini of the H(+)-ATPase that contained either one or two transmembrane segments. Neurospora in vitro translation system supplemented with homologous microsomes was programmed with RNA transcripts of these constructs. When transmembrane segment number one (M1) or number two (M2) of the H(+)-ATPase was engineered into the construct, the resultant protein did not integrate into microsomes. When M1 and M2 were placed in tandem, the resultant protein integrated into microsomes as judged by the criteria of resistance to extraction at pH 11.5 and protection from protease digestion. The integration event depended on ATP and GTP and on microsomal protein(s). We posited that membrane topology of the amino-terminal third of the H(+)-ATPase, and perhaps of other P-type ATPases is achieved by inserting transmembrane segments into membrane in pairs.
粗糙脉孢菌质膜H(+) -ATP酶属于一类称为P型ATP酶的阳离子驱动转运蛋白家族。这些酶的推定跨膜区段含有一个或多个带电荷的残基。确定了带有带电荷残基的跨膜区段整合到其同源膜中的条件。我们构建了融合蛋白，其两侧是H(+) -ATP酶氨基和羧基末端的亲水区段，这些融合蛋白含有一个或两个跨膜区段。用这些构建体的RNA转录本对补充有同源微粒体的粗糙脉孢菌体外翻译系统进行编程。当将H(+) -ATP酶的跨膜区段1（M1）或跨膜区段2（M2）设计到构建体中时，所得蛋白质未整合到微粒体中。当M1和M2串联放置时，根据在pH 11.5下抗提取和免受蛋白酶消化的标准判断，所得蛋白质整合到微粒体中。整合事件取决于ATP、GTP和微粒体蛋白。我们推测，H(+) -ATP酶氨基末端三分之一的膜拓扑结构，可能还有其他P型ATP酶的膜拓扑结构，是通过将跨膜区段成对插入膜中实现的。
