Michaels A, Margulies M M
Biochim Biophys Acta. 1975 May 16;390(3):352-62. doi: 10.1016/0005-2787(75)90356-1.
A system which incorporates amino acids into proteins of chloroplast membranes of Chlamydomonas reinhardti is described. It consists of chloroplast ribosomes bound to thylakoid membranes and cell extract. mRNA is present in this thylakoid-ribosome complex, since neither initiation nor RNA synthesis seems to be required for amino acid incorporation. Incorporation requires ATP, GTP and a soluble portion of cell extract. It is inhibited by chloramphenicol, but not cycloheximide. Most incorporated radioactivity remains bound to the membranes. Although a large portion of this labeled membrane-bound protein occurs as nascent polypeptides, a portion appears at least four products of discrete molecular weights. The major in vitro product migrates as a polypeptide of 23 000 daltons. We conclude that a portion of chloroplast membrane proteins is not only made within the chloroplast, but directly on the membranes. We had previously observed that release of membrane-bound ribosomes is partially dependent on puromycin, and concluded that some membrane-bound ribosomes were attached to the membranes through nascent protein chains. Thus, our results suggest that some chloroplast membrane proteins are inserted into the membranes as they are synthesized. This chloroplast membrane amino acid incorporation system offers a promising tool for studying biosynthesis of membrane proteins, and how they become inserted into chloroplast thylakoids to form functional membranes.
本文描述了一种将氨基酸掺入莱茵衣藻叶绿体膜蛋白中的系统。该系统由结合在类囊体膜上的叶绿体核糖体和细胞提取物组成。由于氨基酸掺入似乎既不需要起始过程也不需要RNA合成，所以mRNA存在于这种类囊体-核糖体复合物中。掺入过程需要ATP、GTP和细胞提取物的可溶部分。它受氯霉素抑制，但不受环己酰亚胺抑制。大多数掺入的放射性物质仍与膜结合。尽管这种标记的膜结合蛋白大部分以新生多肽的形式出现，但有一部分至少呈现出四种分子量不同的产物。体外主要产物迁移为一条23000道尔顿的多肽。我们得出结论，一部分叶绿体膜蛋白不仅在叶绿体内合成，而且直接在膜上合成。我们之前观察到膜结合核糖体的释放部分依赖于嘌呤霉素，并得出结论，一些膜结合核糖体是通过新生蛋白质链附着在膜上的。因此，我们的结果表明，一些叶绿体膜蛋白在合成过程中就插入到膜中。这种叶绿体膜氨基酸掺入系统为研究膜蛋白的生物合成以及它们如何插入叶绿体类囊体以形成功能膜提供了一个很有前景的工具。
