Nilsson R, Brunner J, Hoffman N E, van Wijk K J
Department of Biochemistry, Stockholm University, S-10691 Stockholm, Sweden.
EMBO J. 1999 Feb 1;18(3):733-42. doi: 10.1093/emboj/18.3.733.
The mechanisms of targeting, insertion and assembly of the chloroplast-encoded thylakoid membrane proteins are unknown. In this study, we investigated these mechanisms for the chloroplast-encoded polytopic D1 thylakoid membrane protein, using a homologous translation system isolated from tobacco chloroplasts. Truncated forms of the psbA gene were translated and stable ribosome nascent chain complexes were purified. To probe the interactions with the soluble components of the targeting machinery, we used UV-activatable cross-linkers incorporated at specific positions in the nascent chains, as well as conventional sulfhydryl cross-linkers. With both cross-linking approaches, the D1 ribosome nascent chain was photocross-linked to cpSRP54. cpSRP54 was shown to interact only when the D1 nascent chain was still attached to the ribosome. The interaction was strongly dependent on the length of the nascent chain that emerged from the ribosome, as well as the cross-link position. No interactions with soluble SecA or cpSRP43 were found. These results imply a role for cpSRP54 in D1 biogenesis.
叶绿体编码的类囊体膜蛋白的靶向、插入和组装机制尚不清楚。在本研究中，我们使用从烟草叶绿体中分离的同源翻译系统，研究了叶绿体编码的多聚体D1类囊体膜蛋白的这些机制。对psbA基因的截短形式进行翻译，并纯化稳定的核糖体新生链复合物。为了探究与靶向机制的可溶性成分的相互作用，我们使用了掺入新生链特定位置的紫外线可激活交联剂，以及传统的巯基交联剂。通过这两种交联方法，D1核糖体新生链被光交联到cpSRP54。结果表明，只有当D1新生链仍附着在核糖体上时，cpSRP54才会发生相互作用。这种相互作用强烈依赖于从核糖体中出现的新生链的长度以及交联位置。未发现与可溶性SecA或cpSRP43有相互作用。这些结果表明cpSRP54在D1生物合成中发挥作用。
