In vitro synthesis and membrane insertion of bovine MP26, an integral protein from lens fiber plasma membrane.
In vitro synthesis and membrane insertion of bovine MP26, an integral protein from lens fiber plasma membrane.
复制标题
牛 MP26（一种来自晶状体纤维质膜的整合蛋白）的体外合成和膜插入。
DOI：
10.1083/jcb.96.3.633
复制
发表时间：
1983
期刊：
影响因子：
--
通讯作者：
Goodenough,DA
中科院分区：
文献类型：
--
作者：
Paul,DL;Goodenough,DA
利用寡聚（dt）-纤维素层析法从牛透镜全RNA中富集poly A mRNA，在网织红细胞裂解物系统中指导透镜纤维质膜的主要蛋白质MP 26的合成。通过免疫沉淀富集合成的MP26。体外合成的MP26具有与天然分子难以区分的电泳迁移率。MP26显示了对犬胰腺微粒体的共翻译要求，以便发生膜缔合。微粒体相关的体外合成的MP 26显示出对胰凝乳蛋白酶消化的敏感性，这与分离的透镜纤维质膜中的天然MP 26的敏感性相似，表明MP 26正确插入微粒体中。使用N-甲酰基-[35 S]甲硫氨酰tRNA作为标记物的MP26的合成和膜插入证明没有蛋白水解加工或显著的糖基化伴随膜插入。胰凝乳蛋白酶裂解膜插入，N-甲酰基-[35 S]蛋氨酸标记的MP26导致标签的损失，这表明在体外合成的MP26的N-末端面向细胞质。
Synthesis of MP26, the principal protein of lens fiber plasma membranes, was directed in the reticulocyte lysate system by poly A mRNA enriched from whole bovine lens RNA using oligo (dt)-cellulose chromatography. Synthesized MP26 was enriched by immune precipitation. The in vitro-synthesized MP26 had an electrophoretic mobility indistinguishable from that of the native molecule. MP26 showed a cotranslational requirement for dog pancreas microsomes in order for membrane association to occur. Microsome-associated in vitro-synthesized MP26 showed a sensitivity to digestion with chymotrypsin which was similar to the sensitivity of native MP26 in isolated lens fiber plasma membranes, indicating correct insertion of the MP26 into the microsome. Synthesis and membrane insertion of MP26 using N-formyl-[35S]methionyl tRNA as label demonstrated that no proteolytic processing or significant glycosylation accompanied membrane insertion. Chymotryptic cleavage of membrane-inserted, N-formyl-[35S]methionine-labeled MP26 resulted in loss of label, suggesting that the N-terminal of the in vitro-synthesized MP26 faces the cytoplasm.