Adaptive spatiotemporal organization of chloroplast ATP synthase in Chlamydomonas reinhardtii

莱茵衣藻叶绿体 ATP 合酶的适应性时空组织

• 批准号: 524779207
• 负责人: Dr. Felix Buchert
• 金额: --
• 依托单位: Institut für Integrative Zellbiologie und Physiologie (IIZP)
• 依托单位国家: 德国
• 项目类别: Research Units
• 资助国家: 德国
• 项目状态: 未结题
• 来源: https://gepris.dfg.de/gepris/projekt/524779207?language=en
• 关键词: Adaptive; spatiotemporal; organization; chloroplast; ATP

The thylakoid ultrastructure of the green alga Chlamydomonas reinhardtii provides dynamic sub-compartments where specific alterations in ATP synthase activity and its spatiotemporal organization could occur. ATP synthase is a major bioenergetic complex that affects H+ usage, changes the proton motive force (PMF) and eventually impacts photosynthetic electron transfer regulation. The objective of this project is to dissect the tight relation between photosynthetic and ATP synthase activity and the effect of spatiotemporal features, in particular the dynamic ultrastructure of thylakoids as well as the localization and mobility of ATP synthase within. To do so, ATP synthase will be engineered at different sites with respective sensors for ATP and pH, as part of the Z2 Project. Attaching of fluorescent sensors, or a tag for single molecule labeling, will enable us to trace an individual enzyme and measure local bioenergetics parameters. The tagged enzyme will be monitored under various cellular conditions and, together with its recorded activity, we will try to establish a correlation with a possible change in its molecular organization and/or its sub-compartmental location. Activity will be determined by established time-resolved optical methods and through novel high-resolution ratiometric readouts of fluorescent sensors for pH and ATP. Spatiotemporal organization and changes in the protein environment will be recorded by fluorescence lifetime imaging and single particle tracking in combination with biochemical analyses. An impact on the thylakoid ultrastructure as well as details on the protein complex itself and yet unknown interactors will be gathered via cryogenic electron microscopy and mass spectrometry. Their relevance on the PMF amplitude in the light and in darkness will be tested in a joint effort with other teams of the research unit. The sensor-equipped ATP synthase will also be a valuable tool for other members of the consortium, as it will allow for microscopic quantifications of both ATP synthase substrates – the electrochemical PMF (as pH) and the chemical adenylate status of the cell (as ATP).

绿色藻类莱茵衣藻的类囊体超微结构提供了动态的亚区室，其中ATP合酶活性及其时空组织可能发生特定的改变。ATP合成酶是一种重要的生物能复合物，它影响H+的利用，改变质子动力（PMF），并最终影响光合电子传递调节。本项目的目的是剖析光合作用和ATP合酶活性之间的密切关系和时空特征的影响，特别是类囊体的动态超微结构以及ATP合酶的定位和移动性。为此，作为Z2项目的一部分，ATP合成酶将在不同的地点进行工程改造，并配备ATP和pH传感器。附着荧光传感器或单分子标记标签，将使我们能够追踪单个酶并测量局部生物能量学参数。将在各种细胞条件下监测标记的酶，并与其记录的活性一起，我们将尝试建立与其分子组织和/或其亚隔室位置的可能变化的相关性。活性将通过已建立的时间分辨光学方法和通过pH和ATP荧光传感器的新型高分辨率比率读数来确定。蛋白质环境的时空组织和变化将通过荧光寿命成像和单粒子跟踪结合生化分析来记录。对类囊体超微结构的影响以及蛋白质复合物本身的细节和未知的相互作用将通过低温电子显微镜和质谱法收集。他们的PMF振幅在光明和黑暗中的相关性将与研究单位的其他团队共同努力进行测试。配备传感器的ATP合酶也将是该联盟其他成员的一个有价值的工具，因为它将允许ATP合酶底物的微观定量-电化学PMF（作为pH值）和细胞的化学腺苷酸状态（作为ATP）。