Analysis of factors involved in PSII biogenesis and repair using proteomics and lipidomics
Michael Schroda – Frederik Sommer – Timo Mühlhaus – Sandro Keller
The most important reaction for most life forms on earth is oxygenic photosynthesis. In this process, light energy drives the oxidation of water and the transport of extracted electrons along an electron transport chain embedded in the thylakoid membranes for the generation of NADPH and ATP. Key to this process is photosystem II (PSII), the site of water oxidation. PSII contains 20 protein subunits as well as many cofactors including chlorophylls, pheophytins, β-carotenes, lipids, haem irons, a non-haem iron, manganese and calcium. While the function of PSII is rather well understood, only few of the protein factors that drive its biosynthesis and assembly have been identified and it is unclear how the assembly factors cooperate to achieve this challenging task. The ability of PSII to oxidize water comes along with its vulnerability to oxidative damage, in particular of the D1 core subunit, requiring that PSII is continuously being repaired. Also PSII repair requires a plethora of protein factors that drive the disassembly of PSII (super)complexes, the targeted degradation of damaged D1, its replacement by de novo synthesized D1, and the reassembly of the PSII (super)complexes .
We wish to improve our understanding of how PSII is assembled during de novo biogenesis and how it is repaired following exposure of cells to high light. In particular, we want to identify the protein factors involved and to understand the dynamics in both processes. As the role of thylakoid membrane lipid composition in both processes has yet never been addressed, we wish to establish the methodology of lipidomics for studying PSII assembly and repair which will also involve novel copolymers acting as detergents.
In the recently established Chlamydomonas mutant library CLiP we have identified several mutant lines potentially lacking proteins that have been shown to be involved in PSII biogenesis and repair in higher plants, algae, or cyanobacteria (TERC, Hcf136/Ycf48, PAM68, LPA1/REP27, LPA2, PSB28, MPH1, TEF30). (i) We will generate a 15N and a 13C universal standard from pooled mutants and wild type into which three biological replicates of individual 14N/12C mutants and wild type are spiked [2, 3]. Cells will be exposed to low and high light. Thylakoid membranes are then isolated and analyzed by quantitative mass spectrometry to obtain a picture on the up- and downregulated thylakoid-associated proteins (14N/15N) and thylakoid membrane lipids (12C/13C) in mutants versus wild type . As PSII assembly or repair are blocked at specific points, we expect to identify accumulating assembly and repair factors associated with PSII prior to the blocked step and a lipid fingerprint characteristic for that assembly/repair step. (ii) We will analyze PSII assembly intermediates by BN-PAGE and MS-based complexome profiling. We will also employ copolymers of styrene and maleic acid (SMA) as alternatives to conventional detergents to solubilize thylakoid membranes and analyze them by BN-PAGE and mass spectrometry.
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