Geering K, Meyer D I, Paccolat M P, Kraehenbühl J P, Rossier B C
J Biol Chem. 1985 Apr 25;260(8):5154-60.
Insertion of the alpha- and beta-subunits of amphibian epithelial Na+,K+-ATPase into pancreatic microsomes in cell-free systems was shown to be the same as into membranes of intact cells. The glycoproteic beta-subunit was observed to be cotranslationally inserted into endoplasmic reticulum membranes and to adopt a different pattern of N-linked core and terminal sugars in two different amphibian species. The beta-subunit lacks a cleavable signal sequence but quantitative membrane integration required membrane addition at the start of synthesis. Proteolysis of beta-subunit assembled in vitro indicated a cleavable cytoplasmic domain of about 2000 daltons. The catalytic 98-kilodalton alpha-subunit was also membrane-associated during its synthesis in an alkali-resistant fashion and independent of newly synthesized beta-subunit. In contrast to the beta-subunit, membrane integration of the alpha-subunit was possible as late as a time point in its synthesis which corresponded to about 1/3-1/2 of completion of the nascent chain. A small 34 kDa trypsin-resistant fragment of the alpha-subunit was produced at an early stage of synthesis both in the intact cell and in the cell-free system. These results suggest that membrane insertion of both alpha- and beta-subunit occurs during their synthesis but with a different time course.
在无细胞体系中，两栖动物上皮钠钾ATP酶的α和β亚基插入胰腺微粒体的情况与插入完整细胞膜的情况相同。观察到糖蛋白β亚基在翻译过程中插入内质网膜，并在两种不同的两栖动物物种中呈现出不同的N-连接核心糖和末端糖模式。β亚基缺乏可裂解的信号序列，但定量的膜整合需要在合成开始时添加膜。体外组装的β亚基的蛋白水解表明存在一个约2000道尔顿的可裂解细胞质结构域。催化性的98千道尔顿α亚基在合成过程中也以耐碱方式与膜结合，且不依赖新合成的β亚基。与β亚基不同，α亚基的膜整合最晚可在其合成过程中对应新生链完成约1/3 - 1/2的时间点进行。在完整细胞和无细胞体系中，α亚基在合成早期都会产生一个34千道尔顿的抗胰蛋白酶小片段。这些结果表明，α和β亚基的膜插入都发生在它们的合成过程中，但时间进程不同。
