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

类囊体膜的光合作用复合体的一个有趣的特征是它们含有大量的低分子(LMW)蛋白质，这些蛋白质是核和叶绿体编码的。在这里，我们研究了组装因子和单个LMW蛋白在光系统(PS)II的生物发生、光吸收、电子传递过程、磷酸化、稳定和修复中的不同功能，主要是使用相应的核基因和叶绿体基因的敲除。此外，突变谱系将用于鉴定组装过程中出现的亚复合体的组成以及它们在类囊体膜系统中的定位，方法包括天然凝胶、免疫共沉淀(Co-IP)、层析、交联、标记蛋白质、分级技术和质谱学。我们提供了明确的证据，证明LMW膜蛋白PsbN，到目前为止被注释为PSII亚单位，不是这个复合体的一部分，但在组装其异二聚体反应中心方面是必不可少的。将确定PsbN和与PsbN相关的几个前复合体中的蛋白质的直接相互作用伙伴，已获得的相互作用因素将通过分裂泛素分析、蛋白质重叠分析、SPR光谱分析、PsbN标记版本的共IP和质谱学来验证和分析。这些问题有助于我们理解PSII的逐步组装、光捕获、电子在PSII内和向外的传递、水的分解和放氧、膜动力学以及敏感的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.