Role of thylakoid FtsH proteases in biogenesis and degradation of the chloroplast proteins in the photosynthetic model-organism Chlamydomonas reinhardtii – FtsH-Thyl-Chlamy

Taking advantage of the fact that Chlamydomonas is a unicellular, haploid, facultative phototroph and that many photosynthesis mutants are available, we will exploit the possibilities to generate and combine chloroplast and nuclear mutants in Chlamydomonas. Our laboratory has constituted a collection of photosynthesis mutants of Chlamydomonas (http://chlamystation.free.fr/) and has developed an exceptional spectroscopic instrumentation to study photosynthesis by in vivo measurements by time-resolved absorption spectroscopy and fluorescence. Our complementary approaches including genetics, molecular biology, biochemistry and biophysics allow the in depth characterization in vivo and in vitro of photosynthetic complexes.

To address the question of the role of chloroplast proteases in the biogenesis and maintenance of photosynthetic membranes proteins, we chose Chlamydomonas as a photosynthetic model organism to develop a genetic suppressor screen aimed at the identification of proteases involved in the quality control of the cytochrome b6f complex. We have provided the first evidence that a fully green mutant of the chloroplast-located and thylakoid-based ATP-dependent FtsH metalloprotease controls both photosystem II (PSII) and cytochrome b6f recycling upon light and nutrient stress. We used the highly photosensitive phenotype of this mutant for map-based cloning and molecular identification of the suppressor mutation. Mutation ftsH1-R420C is a substitution of an arginine known to be essential for ATPase and protease activity of FtsH in Escherichia coli. Complementation of this mutation by wild-type genomic FTSH1 DNA restores a wild-type non-photosensitive phenotype. We have demonstrated that misassembled cytochrome b6f complexes are degraded through an FtsH-dependent pathway. We have shown that this FtsH mutant does not recycle PSII upon phosphate or sulphur deprivation and provide evidence for a combined role of FtsH and putative Deg proteases in the degradation of the D1 protein from PSII in photoinhibitory conditions and during other related environmental changes. We have provided the first evidence for a light-independent yet FtsH-mediated degradation of PSII under sulphur deprivation in Chlamydomonas reinhardtii.

We propose a basic study of the role of the thylakoid FtsH proteases in the biogenesis and degradation of chloroplast proteins in the photosynthetic model-organism Chlamydomonas reinhardtii. This study will allow the identification of FtsH substrates, the characterization of the regulatory functions of thylakoid FtsH proteases and the availability of protease mutants as versatile tools.
Chloroplast of microalgae offers great promise for the production of proteins of pharmaceutical interest as for the development of novel biofuels. Our mutants with limited proteolytic activity in the chloroplast could be future tools for engineering original production systems.

- Sarah Estelle Pousset. Internship report of Master 2 of UPMC, defended on June 20, 2013. Role of the thylakoid FtsH protease in the remodelling of chloroplast bioenergetics upon sulphur starvation in Chlamydomonas reinhardtii.
- Alizée Malnoë et al. The FtsH Protease of Chlamydomonas contributes to Photosystem II and Cytochrome b6f Remodelling in Stress Conditions. In preparation.

The role of thylakoid FtsH proteases in biogenesis and degradation of chloroplast proteins will be studied in the photosynthetic model-organism Chlamydomonas reinhardtii. We will analyze FtsH mutants and double mutants combining defects in FtsH proteolytic activity and in other biogenesis or proteolytic processes. Two different types of FtsH proteases are found in thylakoid membranes and these two types contain several isoforms in Arabidopsis. The situation in Chlamydomonas is much simpler in this respect as both types have only one representative. We have already isolated different allele mutants of FtsH1 and will generate FtsH2 mutants. Stresses are relevant conditions to shine light on the roles of protease and to identify their substrates. As FtsH1 and FtsH2 are thylakoid membrane anchored proteases, we will first test transmembrane photosynthetic complexes as substrates in various stress conditions. In addition to providing insights into the role of proteases in the overall biogenesis process, we have shown that these mutants can rescue partially assembled protein complexes that would otherwise be degraded due to their high protease sensitivity. Thus FtsH mutants are versatile tools for understanding the assembly and function of the photosynthetic apparatus. Taking advantage of the fact that Chlamydomonas is a unicellular, haploid, facultative phototroph and that many photosynthesis mutants are available, we will exploit the possibilities to generate and combine chloroplast and nuclear mutants in Chlamydomonas.