Transport and insertion mechanisms of plastid-encoded thylakoid membrane proteins

叶绿体类囊体膜光合蛋白复合物的生物发生需要高度特异性的蛋白质分选、整合和组装机制以及质体编码亚基。光系统II （PS II）生物发生的核心步骤是编码D1蛋白的质体共翻译插入到类囊体膜中，并随后组装成功能的PS II。我们最近建立了一种技术，利用来自豌豆叶绿体的同源离体翻译系统部分重建共翻译插入的D1。纯化的类囊体膜相关核糖体上升链复合物的免疫学分析和体外插入实验揭示了cpSec/Alb3转位酶和Vipp1在D1生物发生中的作用。此外，我们最近发现，拟南芥基因敲除突变体在D1插入上存在严重缺陷，这是由于翻译核糖体与类囊体膜的结合受损造成的。本研究的目的是：(1)鉴定参与类囊体膜共翻译蛋白插入的新组分，(2)剖析新生D1链在翻译和插入过程中的蛋白质接触，(3)深入了解核糖体上升链复合物靶向和附着于类囊体膜的机制。

The biogenesis of photosynthetic protein complexes of the chloroplast thylakoid membrane requires highly specific protein sorting, integration and assembly mechanisms of nucleus as well as plastid encoded subunits. Central steps in the biogenesis of photosystem II (PS II) are the cotranslational insertion of the plastid encoded D1 protein into the thylakoid membrane and its subsequent assembly into functional PS II. We recently established a technique to partially reconstitute the cotranslational insertion of D1 using a homologous in vitro translation system derived from pea chloroplasts. Immunological analyses of purified thylakoid membrane associated ribosomenascent chain complexes and in vitro insertion experiments revealed a role of the cpSec/Alb3 translocase and Vipp1 in D1 biogenesis. Furthermore, we recently showed that the cpftsy Arabidopsis thaliana knock-out mutant exhibits a severe defect in D1 insertion which is caused by an impaired binding of translating ribosomes to the thylakoid membrane. The aims of this proposal are (I) to identify novel components involved in cotranslational protein insertion in thylakoid membranes, (II) to dissect the protein contacts of the nascent D1 chain during translation and insertion and (III) to get insight into the mechanisms underlying targeting and attachment of ribosomenascent chain complexes to the thylakoid membrane.