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首页> 外文会议>NATO Advanced Research Workshop on The Chloroplast: From Molecular Biology to Biotechnology Kolymbari-Chania, Crete, Greece 10-15 August 1998 >Reconstitution of co-translational targeting of polytopic membrane proteins to the thylakoids in a homologous chloroplast translation system
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Reconstitution of co-translational targeting of polytopic membrane proteins to the thylakoids in a homologous chloroplast translation system

机译：在同源叶绿体翻译系统中重组翻译膜蛋白对类囊体的共翻译靶向

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摘要

Chloroplasts in higher plants posses circular DNA containing 35 genes encoding for essential proteins located in the thylakoid membranes. The mechanisms of targeting and insertion of these proteins are unknown, despite their importance. Based on a number of observations in chloroplasts, it can be postulated that targeting and insertion of the polytoic chloroplast-encoded membrane proteins occurs co-translationally (1-3). Thus in order to reconstitute this targeting and insertion process, a homologous chloroplast in vitro initiation/translation system is required in which plasmid derived transcripts can be faithfully translated. The recent discovery of a translation system isolated from tobacco chloroplasts has opened up novel possibilities to address these important processes at a molecular level. In this paper, we have set out to evaluate the interaction of soluble stromal components cpSRP54, cpSRP43 and SecA with the chloroplast encoded D1 protein, using this translation system. We show that ribosome D1 nascent chain complexes (D1 rncs) can be targeted very efficiently to the membrane and make functional interactions. In addition we show that cpRP54 interacts specifically with D1 rncs of defined length, implying a role for cpSRP54 in D1 biogenesis. To study the extent of conservation and mechanisms of the chloroplast targeting mechanisms with prokaryotes, we translated E. coli Leader peptidase (Lep) in the chloroplast system and attempted targeting Lep to the thylakoid membrane. Lep has two membrane spans and has served as a model protein to study targeting and insertion to the E. coli inner membrane (8-10) as well as to ER membranes (e.g. aa). Lep is made without a cleavable signal sequence and its N- and C-terminal domains face the periplasmic side of the inner membrane (Fig. 4). In E.coli, insertion of Lep is SecA, SecY and SRP dependent (8-10).
机译：高等植物的叶绿体具有环状DNA，该环状DNA包含35个编码类囊体膜中必需蛋白质的基因。尽管它们的重要性，但靶向和插入这些蛋白的机制尚不清楚。基于对叶绿体的大量观察，可以推测多聚叶绿体编码的膜蛋白的靶向和插入是共翻译发生的（1-3）。因此，为了重构该靶向和插入过程，需要同源的叶绿体体外起始/翻译系统，在该系统中可以忠实翻译质粒衍生的转录本。从烟草叶绿体分离的翻译系统的最新发现为在分子水平上解决这些重要过程提供了新的可能性。在本文中，我们已着手使用此翻译系统评估可溶性基质成分cpSRP54，cpSRP43和SecA与叶绿体编码的D1蛋白的相互作用。我们显示，核糖体D1新生链复合物（D1 rncs）可以非常有效地靶向膜并进行功能相互作用。此外，我们显示cpRP54与定义长度的D1 rncs特异性相互作用，暗示cpSRP54在D1生物发生中的作用。为了研究原核生物的保守程度和叶绿体靶向机制的机制，我们在叶绿体系统中翻译了大肠杆菌前导肽酶（Lep），并尝试将Lep靶向类囊体膜。 Lep具有两个跨膜跨距，并已作为模型蛋白来研究靶向和插入大肠杆菌内膜（8-10）以及内质网膜（例如aa）。产生的Lep没有可裂解的信号序列，其N和C末端结构域面对内膜的周质侧（图4）。在大肠杆菌中，Lep的插入取决于SecA，SecY和SRP（8-10）。

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