Levy R, Wiedmann M, Kreibich G
Department of Cell Biology, New York University School of Medicine, New York, NY 10016, USA.
J Biol Chem. 2001 Jan 26;276(4):2340-6. doi: 10.1074/jbc.M004867200. Epub 2000 Oct 17.
The Sec61p complex forms the core element of the protein translocation complex (translocon) in the rough endoplasmic reticulum (rough ER) membrane. Translating or nontranslating ribosomes bind with high affinity to ER membranes that have been stripped of ribosomes or to liposomes containing purified Sec61p. Here we present evidence that the beta subunit of the complex (Sec61beta) makes contact with nontranslating ribosomes. A fusion protein containing the Sec61beta cytoplasmic domain (Sec61beta(c)) prevents the binding of ribosomes to stripped ER-derived membranes and also binds to ribosomes directly with an affinity close to the affinity of ribosomes for stripped ER-derived membranes. The ribosome binding activity of Sec61beta(c), like that of native ER membranes, is sensitive to high salt concentrations and is not based on an unspecific charge-dependent interaction of the relatively basic Sec61beta(c) domain with ribosomal RNA. Like stripped ER membranes, the Sec61beta(c) sequence binds to large ribosomal subunits in preference over small subunits. Previous studies have shown that Sec61beta is inessential for ribosome binding and protein translocation, but translocation is impaired by the absence of Sec61beta, and it has been proposed that Sec61beta assists in the insertion of nascent proteins into the translocation pore. Our results suggest a physical interaction of the ribosome itself with Sec61beta; this may normally occur alongside interactions between the ribosome and other elements of Sec61p, or it may represent one stage in a temporal sequence of binding.
Sec61p复合物构成了糙面内质网（糙面内质网）膜中蛋白质转运复合物（转运体）的核心元件。正在翻译或未翻译的核糖体以高亲和力与已去除核糖体的内质网膜或含有纯化Sec61p的脂质体结合。在此我们提供证据表明该复合物的β亚基（Sec61β）与未翻译的核糖体发生接触。一种含有Sec61β胞质结构域（Sec61β(c)）的融合蛋白可阻止核糖体与去除核糖体的内质网衍生膜结合，并且还以接近核糖体与去除核糖体的内质网衍生膜的亲和力直接与核糖体结合。Sec61β(c)的核糖体结合活性与天然内质网膜一样，对高盐浓度敏感，且并非基于相对碱性的Sec61β(c)结构域与核糖体RNA的非特异性电荷依赖性相互作用。与去除核糖体的内质网膜一样，Sec61β(c)序列优先与大核糖体亚基结合而非小亚基。先前的研究表明Sec61β对于核糖体结合和蛋白质转运并非必需，但Sec61β的缺失会损害转运，并且有人提出Sec61β有助于新生蛋白质插入转运孔。我们的结果表明核糖体自身与Sec61β存在物理相互作用；这可能通常与核糖体和Sec61p的其他元件之间的相互作用同时发生，或者它可能代表结合时间序列中的一个阶段。
