Biochemical characterization of membrane complexes of the thylakoidal Delta pH-dependent twin arginine translocase (Tat translocase)

在叶绿体类囊体膜上的四条蛋白质转运途径中，DpH/达特途径是唯一的，它能转运折叠蛋白质，并能提供光合作用产生的跨膜质子梯度。从DpH/达特机制的三个组分（TatA、TatB、TatC）中，迄今为止在异聚复合物中仅发现了TatB和TatC。本研究拟通过以下方法对DpH/达特转位酶进行表征：（1）利用单链抗体（scFv）亲和层析法从类囊体膜上分离TatB/C和TatA复合物。(2)通过不同的色谱方法和质谱法分离单个达特蛋白，以鉴定可能的修饰。(3)通过亲和色谱法分离达特复合物的假定配体。(4)使用含EGFP的嵌合前体蛋白评估DpH/达特易位酶对底物折叠状态的潜在特异性。(5)使用特定工程嵌合底物体外捕获活性构象的转位酶。(6)通过这种嵌合底物的瞬时表达在体内捕获活性易位酶。

Among the four protein transport pathways operating at the chloroplast thylakoid membrane, the DpH/Tat pathway is unique with respect to its ability to transport folded proteins and its energy source, the transmembrane proton gradient generated upon photosynthesis. From the three components of the DpH/Tat machinery (TatA, TatB, TatC), only TatB and TatC have so far been found in heteromeric complexes. We intend to characterize the DpH/Tat translocase by following approaches: (1) Isolation of the TatB/C and TatA complexes from thylakoid membranes using affinity chromatography based on single chain antibody fragments (scFv). (2) Isolation of individual Tat proteins by different chromatographic methods and mass spectrometry in order to identify possible modifications. (3) Isolation of presumed ligands of the Tat complexes by affinity chromatography. (4) Assessing a potential specificity of the DpH/Tat translocase for the folding state of the substrates using EGFP-containing chimeric precursor proteins. (5) In vitro trapping of the translocase in its active conformation using specifically engineered chimeric substrates. (6) In vivo trapping of the active translocase by transient expression of such chimeric substrates.