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Rubisco Biogenesis and Assembly in Chlamydomonas Reinhardtii Wojciech Wietrzynski

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Rubisco Biogenesis and Assembly in Chlamydomonas Reinhardtii Wojciech Wietrzynski Rubisco biogenesis and assembly in Chlamydomonas reinhardtii Wojciech Wietrzynski To cite this version: Wojciech Wietrzynski. Rubisco biogenesis and assembly in Chlamydomonas reinhardtii. Molecular biology. Université Pierre et Marie Curie - Paris VI, 2017. English. NNT : 2017PA066336. tel- 01770412 HAL Id: tel-01770412 https://tel.archives-ouvertes.fr/tel-01770412 Submitted on 19 Apr 2018 HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. PhD Thesis of the Pierre and Marie Curie University (UPMC) Prepared in the Laboratory of Molecular and Membrane Physiology of the Chloroplast, UMR7141, CNRS/UPMC Doctoral school: Life Science Complexity, ED515 Presented by Wojciech Wietrzynski for the grade of Doctor of the Pierre and Marie Curie University Rubisco biogenesis and assembly in Chlamydomonas reinhardtii Defended on the 17th of October 2017 at the Institut of Physico-Chemical Biology in Paris, France Phd Jury: Angela Falciatore, CNRS/UPMC, president Michel Goldschmidt-Clermont, Univeristy of Geneva, reviewer Michael Schroda, University of Kaiserslautern, reviewer Cecile Raynaud, CNRS/IPS2, examinator Steven Ball, University of Lille, examinator Francis-André Wollman, CNRS/UPMC, supervisor Katia Wostrikoff, CNRS/UPMC, supervisor 2 Acknowledgements You all already know that I’m not very good at it but I’ll try… Thanks to Mama and Papa for always letting me choose whatever I want to do, going to France included; To Francis-André for letting me stay in France in his laboratory, but foremost for all advices and critical comments and for making the lab above all, a place of discussion (not only scientific); The biggest thanks to Katia for having me work with her for those 4 years! For defending me, and in general being patient despite me being difficult at times (most of the time); Thanks to all that shared the bureau with me: Loreto, Han-yi, Domitille, Benjamin, Sandrine and the Ficus. Especially, to Sheriff Sandrine for keeping me in line and for all the food she shared with me! I’d like to thank all the members: past and present of Franics-André’s lab, everyone was special and memorable. I would be too long to evoke all the good memories I have with you. Just a special mention to The Axis: Stefania, Marina and Felix for trying to win this time. And Stephan, especially at the beginning, for always having time for a beer and talk; To follow, thanks to all the colleagues I have found in the Institute: Marcello, Max, Justin and all the others; Thanks to my friends Larissa and Wojtek for all the support. Wojtek in particular, for being my adversary for such a long time! And last but not least, for my special little cat, Nathalie: for teaching me French, how to take care of cats, how to be more social and for being there for me! It was a pleasure. 3 Table of contents Abbreviations 8 Opening words 9 1. General Introduction 11 1.1. Part I - Photosynthesis, endosymbiosis and consequences 11 1.1.1. Chloroplasts-a specialized organelle for oxygenic photosynthesis 11 1.1.2. Chloroplast genome organization 13 1.1.3. Endosymbiotic gene transfer: facts and hypotheses 15 1.1.4. Protein rerouting to the chloroplast 17 1.2. Part II Nucleus and chloroplast crosstalk 18 1.2.1. Chloroplast transcription and translation apparatus and regulation 19 1.2.1.1. Transcription in chloroplast 19 1.2.1.2. mRNA degradation in chloroplast 19 1.2.1.3. Translation machinery 20 1.2.1.4. Organization of translation 21 1.2.2. Nucleus-encoded regulators of chloroplast gene expression 22 1.2.2.1. TPR 22 1.2.2.2. PPR 23 1.2.2.3. OPR 24 1.2.2.4. mTERF 25 1.2.2.5. Convergent evolution- convergent role? 26 1.2.2.6. OTAFs in chloroplast 27 1.2.3. Retrograde signaling 28 1.3. Part III: Regulatory processes involved in photosynthetic complex assembly 31 1.3.1. Concerted accumulation of subunits 31 1.3.2. Proteolysis 31 1.3.3. The CES process 32 1.3.3.1. Mechanism 33 1.3.3.2. Special case of ATPsynthase 35 1.3.3.3. CES as a regulatory process 36 1.3.3.4. CES in higher plants 36 1.3.3.5. CES and anterograde regulation 37 1.3.3.6. Additional remarks 38 2. Rubisco 39 2.1. Rubisco evolution and clades 39 4 2.2. Subunits and structure (Rubisco Type I) 40 2.3. Rubisco reaction 43 2.3.1. Oxygenation 44 2.3.2. Unproductivity 45 2.3.3. Efficiency 45 2.4. Rubisco biogenesis 46 2.4.1. Expression and regulation 46 2.4.2. Folding and assembly 51 2.4.2.1. CPN60 complex 52 2.4.2.2. BSD2 53 2.4.2.3. Rubisco assembly chaperones 53 2.4.2.4. RBCX 54 2.4.2.5. RAF1 56 2.4.2.6. Comparison 58 2.4.2.7. RAF2 58 2.4.2.8. SSU folding 60 3. Interlude 60 4. Chapter I : MRL1 role in rbcL translation regulation 62 4.1. Introduction 62 4.2. Part I - Searching for new OTAFs involved in rbcL gene expression 63 4.2.1. Genetic approach: Mutagenesis 63 4.2.1.1. Negative selection screen 63 4.2.1.2. rbcL regulatory sequence drives cytosine deaminase expression 65 4.2.1.3. Mutagenesis of CRE1 66 4.2.2. Biochemical approach: Co-immunoprecipitation of MRL-HA 68 4.2.2.1. Generation of a tagged MRL1 strain 68 4.2.2.2. ImmunoPrecipitation and Mass-spectrometry 70 4.3. Part II - Is MRL1 alone? 72 4.3.1. MRL1 level limits rbcL mRNA accumulation and is required for LSU 72 4.3.2. MRL1 is a stable protein 73 4.3.3. MRL1 is required for rbcL translation 74 4.4. Discussion and perspectives 77 4.4.1. In a search of rbcL T-factor 77 4.4.2. M-factor’s dual function? 79 4.4.3. Insight in MRL1 mode of action? 79 4.4.4. MRL1, a regulator of Rubisco accumulation? 80 5 5. Chapter II - Rubisco LSU synthesis depends on its oligomerization state in Chlamydomonas reinhardtii 82 5.1. Abstract 83 5.2. Introduction 84 5.3. Results 86 5.3.1. rbcL downregulation of synthesis in Chlamydomonas RBCS mutant. 86 5.3.2. LSU initiation of translation is impaired in absence of SSU 86 5.3.3. Translation initiation is inhibited by unassembled LSU 87 5.3.4. Assembly intermediates accumulate in absence of SSU 88 5.3.5. CES regulation no longer occurs in Rubisco oligomerization mutants 89 5.3.6. Rubisco assembly intermediates in the LSU8mut oligomerization mutant reveal the LSU CES repressor 91 5.4. Discussion 92 5.4.1. LSU CES results from a negative autoregulation on translation initiation 92 5.4.2. Fate of unassembled LSU 94 5.4.3. Insights into Rubisco assembly pathway 94 5.4.4. Tentative identification of the repressor form 96 5.4.5. Evolutionary conservation of Rubisco CES process 97 5.5. Materials and Methods 98 5.6. Figure legends 101 5.6.1. Fig. 1 LSU synthesis rate and accumulation in absence of its assembly partner 101 5.6.2. Fig. 2 Swapping of rbcL regulatory sequences impairs the CES regulation 101 5.6.3. Fig. 3 Expression of cyt. f is inhibited in the absence of Rubisco small subunit 101 5.6.4. Fig. 4 CES regulation no longer occurs in the absence of LSU accumulation 102 5.6.5. Fig. 5 LSU assembly intermediates accumulate in the SSU-lacking strain 102 5.6.6. Fig. 6 LSU2 mutations alter LSU accumulation and CES regulation 102 5.6.7. Fig. 7 Disruption of LSU oligomerization alters LSU CES regulation 103 5.6.8. Sup. Fig. 1 Anti-RAF1 antibody 103 5.6.9. Sup. Fig. 2 RAF1 accumulates in the absence of LSU 103 5.6.10. Sup. Fig. 3 Close-up of the mutated residues in LSU structure 103 5.7. Bibliography 104 5.8. Figure list 107 6. Chapter III - Further exploration of limitations in Rubisco biosynthesis – Supplementary results and discussion 113 6.1. Additional results for: “Rubisco LSU synthesis depends on its oligomerization state in Chlamydomonas reinhardtii” 114 6 6.1.1. Generation of ΔRBCS;5’UTRpsaA:rbcL strain and its additional phenotype 114 6.1.2. Reporter gene accumulation is altered in ΔRBCS;5’UTRpsaA:rbcL 115 6.1.3. What happens with LSU when Rubisco assembly is perturbed? 117 6.1.4. Stability of LSU 117 6.1.5. Fractionation 118 6.1.6. EPYC1 accumulation is decreased in Rubisco deficient strains 121 6.1.7. Quantification of LSU and RAF1 levels 121 6.2. Discussion 124 6.2.1. LSU is relatively stable when chaperone-bound 124 6.2.2. Limiting steps of LSU assembly 124 6.2.3. Mutated LSU is directed to aggregates 125 6.2.4. EPYC1 accumulates in coordination with Rubisco 126 6.2.5. Conclusions from LSU and RAF1 quantifications 127 6.3. Additional comments 127 7. General discussion 129 8. Conclusions 134 9. Materials 135 10. Methods 137 10.1. Cultures 137 10.2.
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