Project Details
Projekt
Adaptive spatiotemporal organization of chloroplast ATP synthase in Chlamydomonas reinhardtii
Applicants
Dr. Felix Buchert; Professorin Dr. Karin Busch
Subject Area
Plant Biochemistry and Biophysics
Term
since 2023
Project identifier
Deutsche Forschungsgemeinschaft (DFG) - Project number 507704013
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).
DFG Programme
Research Units
