Tam L Y, Loo T W, Clarke D M, Reithmeier R A
Department of Medicine, University of Toronto, Ontario, Canada.
J Biol Chem. 1994 Dec 23;269(51):32542-50.
The insertion of Band 3, the human erythrocyte anion exchanger, into microsomal membranes was studied in an in vitro reticulocyte lysate translation system. Band 3 consists of a 43-kDa amino-terminal cytosolic domain and a carboxyl-terminal 52-kDa membrane domain containing up to 14 transmembrane segments with a single N-glycosylation site at Asn-642. Insertion of truncated Band 3 molecules into microsomal membranes was assayed by glycosylation, resistance to alkaline extraction, and tryptic removal of the cytosolic domain. Truncations containing either the first four or the last eight putative transmembrane segments were stably integrated into microsomes showing that an intact membrane domain was not required for membrane integration. Furthermore, the extracytosolic domain following the seventh transmembrane segment was properly translocated across the microsomal membrane and glycosylated whether the seventh transmembrane segment was the first, last, or the only transmembrane segment in the construct. The ability of the entire membrane domain, the truncated domain beginning with the seventh transmembrane segment, or the seventh transmembrane segment to insert into microsomes was dependent on the presence of the signal recognition particle receptor. The seventh transmembrane segment in Band 3 therefore has the topogenic properties of an internal signal sequence.
在体外网织红细胞裂解物翻译系统中研究了人类红细胞阴离子交换蛋白3（Band 3）插入微粒体膜的过程。Band 3由一个43 kDa的氨基末端胞质结构域和一个羧基末端52 kDa的膜结构域组成，该膜结构域包含多达14个跨膜片段，在Asn-642处有一个单一的N-糖基化位点。通过糖基化、对碱性提取的抗性以及胰蛋白酶去除胞质结构域来检测截短的Band 3分子插入微粒体膜的情况。包含前四个或最后八个假定跨膜片段的截短片段稳定地整合到微粒体中，这表明膜整合不需要完整的膜结构域。此外，无论第七个跨膜片段是构建体中的第一个、最后一个还是唯一的跨膜片段，第七个跨膜片段之后的胞外结构域都能正确地跨微粒体膜转运并进行糖基化。整个膜结构域、从第七个跨膜片段开始的截短结构域或第七个跨膜片段插入微粒体的能力取决于信号识别颗粒受体的存在。因此，Band 3中的第七个跨膜片段具有内部信号序列的拓扑性质。
