拟南芥BFA1蛋白调控叶绿体ATP合酶组装的分子机理研究

The chloroplast ATP synthase is a key enzyme in energy conversion in photosynthetic organisms. Rapid and efficient biogenesis of chloroplast ATP synthase is required for growth and development as well as photosynthesis of photosynthetic organisms. The chloroplast ATP synthase is composed of nine different subunits encoded by nuclear and plasmid genes with a different stoichiometry. However, our knowledge of molecular mechanisms of efficient biogenesis of chloroplast ATP synthase is still limited. By forward genetics, we have isolated an Arabidopsis mutant bfa1 which is defective in the accumulation of ATP synthase. It is showed that BFA1 is a stromal protein with unknown function and assembly of the chloroplast ATP synthase was less efficient in bfa1 than in the wild type. To investigate the molecular mechanism of BFA1 in assembly of the chloroplast ATP synthase, the following research objectives and strategies will be used in this work: 1) To confirm which stage of chloroplast ATP synthase assembly was affected in the mutants by protein labeling and chase experiments. 2) To identify the protein interacting with BFA1 by Co-IP and yeast two hybridization system and determine the interacting amino acid residues with BFA1 by crosslinking in combination with mass spectrometry. 3) To analyze protein structure of BFA1 by crystals technology and to simulate interaction between BFA1 and its interaction protein via molecular docking. The results will finally dissect the molecular function of BFA1 in chloroplast ATP synthase assembly and provide new insights into the assembly process of chloroplast ATP synthase.

叶绿体ATP合酶是光合生物能量代谢的关键酶，快速、高效地生成叶绿体ATP合酶复合物是光合生物进行光合作用和生长发育的重要保证。由于叶绿体ATP合酶的组成和结构复杂，其高效组装的分子机制还不完全清楚。我们采用正向遗传学策略筛选获得了一个叶绿体ATP合酶含量特异性降低的拟南芥突变体bfa1，前期分析结果表明其突变基因编码一个功能未知的叶绿体基质蛋白，缺失此蛋白的bfa1突变体中叶绿体ATP合酶的组装受到抑制。本项目拟采用蛋白质标记-追踪技术研究突变体bfa1中叶绿体ATP合酶组装受阻的阶段；利用Co-IP和酵母双杂交技术以及蛋白质交联结合质谱分析检测BFA1的互作蛋白和互作关键位点；通过蛋白质晶体技术解析BFA1蛋白结构，根据晶体结构和互作位点模拟BFA1与底物亚基的分子对接，阐明BFA1调控叶绿体ATP合酶组装的分子机理。相关结果对于深入理解叶绿体ATP合酶组装过程具有重要的科学意义。

叶绿体ATP合酶是光合生物能量代谢的关键酶，但是其高效组装的分子机制还不完全清楚。我们鉴定获得了叶绿体ATP合酶组装因子BFA1，蛋白质标记-追踪实验结果表明bfa1中叶绿体ATP合酶CF1亚复合物组装受阻；利用Co-IP和酵母双杂交技术鉴定了BFA1与CF1β和γ亚基的互作关键位点；利用蛋白质晶体技术解析了BFA1蛋白的结构；通过计算机分子对接分析发现BFA1和CF1β/γ能够通过互作关键位点对接且没有空间冲突。我们提出了BFA1作为支架蛋白促进CF1α/β异源二聚体与CF1γ结合的工作模型，后续其他CF1α/β异源二聚体的组装可能会使CF1γ亚基发生位移，从而完成CF1亚复合物的组装。相关结果对于深入理解叶绿体ATP合酶组装过程具有重要的科学意义。