Function of thylakoid low molecular weight proteins and assembly factors in the biogenesis, activity and stability of photosynthetic complexes

类囊体膜的光合复合体的一个有趣的特征是它们大量的低分子量（LMW）蛋白质，这些蛋白质是核和质体编码的。本文研究了装配因子和LMW蛋白在拟南芥和烟草的生物发生、光吸收、电子传递过程、磷酸化、稳定和主要光系统（PS）II修复中的独特功能，分别使用了相应的核和质体基因敲除。此外，突变库将用于鉴定组装过程中出现的亚复合物的组成及其在类囊体系统中的定位，使用天然凝胶，共免疫沉淀（Co-IP），色谱，交联，标记蛋白，分离技术和质谱。我们提供了明确的证据，证明LMW膜蛋白PsbN，迄今为止被注释为PSII亚基，不是该复合体的一部分，而是其异二聚体反应中心组装所必需的。将鉴定PsbN的直接相互作用伙伴和与PsbN相关的几个前体中的蛋白质，已经获得的相互作用因子将使用分裂泛素测定、蛋白质覆盖测定、SPR光谱测定、带有标记版本的PsbN的Co-IP和质谱测定进行验证和分析。所解决的问题有助于我们理解PSII的逐步组装、光捕获、电子内外转移、水分裂和O2演化、膜动力学以及易感PSII从光抑制和热失活中恢复。

An intriguing feature of the photosynthetic complexes of the thylakoid membrane is their high number of low molecular weight (LMW) proteins, which are nuclear and plastid encoded. Here we investigate the distinct function of assembly factors and of individual LMW proteins in the biogenesis, light absorbance, electron transport processes, phosphorylation, stabilisation and repair mainly of photosystem (PS)II using available knockouts of corresponding nuclear and plastid genes in Arabidopsis and tobacco, respectively. Furthermore, the mutant repertoire will be used to identify the composition of subcomplexes appearing during assembly and their localisation within the thylakoid system using native gels, co-immunoprecipitation (Co-IP), chromatography, cross-linking, tagged proteins, fractionation techniques and mass spectrometry. We provide clear evidence that the LMW membrane protein PsbN, which hitherto was annotated as PSII subunit, is not part of this complex but essentially required for assembly of its heterodimeric reaction centre. Direct interaction partners of PsbN and proteins associated in several precomplexes together with PsbN will be identified, already obtained interacting factors will be verified and analysed using split-ubiquitin assays, protein overlay assays, SPR spectrometry, Co-IP with tagged versions of PsbN and mass spectrometry. The addressed issues assist in our understanding of the stepwise assembly, light capture, transfer of electrons within and outgoing of PSII, water splitting and O2 evolution, membrane dynamics and recovery of the susceptible PSII from photoinhibition and heat inactivation.