The Biogenesis of Photosynthetic Complexes PSII and b6f DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Sara Guenthner Cline Graduate Program in Plant Cellular and Molecular Biology The Ohio State University 2012 Dissertation Committee: Hamel, Patrice, Advisor Bird, Amanda Mackey, Dave Somers, Dave Copyright by Sara Guenthner Cline 2012 Abstract Photosynthetic complex assembly within the plastid is a blossoming field as science uncovers additional requirements and levels of regulation in the production of these protein complexes. Using both forward and reverse genetics, our lab has revealed two new players in the assembly of Photosystem II and the b6f complex. The first of these factors is LTO1, identified as the thiol oxidant for the production of a disulfide bond within PsbO, a PSII luminal protein. The second is an OPR protein with a novel role in the attachment of heme to cytochrome f of the b6f complex. The presence of these assembly factors opens exciting new doors in the possibilities of plastid b, as demonstrated by our relocation of the nuclear ARG9 gene into the plastid genome. ii Dedicated to my Dad. iii Acknowledgments Here I would like to thank my P.I. for his patience and my lab mates for their help. iv Vita 2001................................................................Trimble County High School 2005................................................................B.A. Biology, Transylvania University, 2005 to present ..............................................Graduate Teaching Associate, Department of Plant Cellular and Molecular Biology, The Ohio State University Publications Mohamed Karamoko, Sara Cline, Kevin Redding, Natividad Ruiz, and Patrice Hamel. Lumen Thiol Oxidoreductase1, a Disulfide Bond-Forming Catalyst, Is Required for the Assembly of Photosystem II in Arabidopsis. Plant Cell (2011) 28 (12): 4462-4475. Claire Remacle, Sara Cline, Layla Boutaffala, Stéphane Gabilly, Véronique Larosa, M. Rosario Barbieri, Nadine Coosemans, and Patrice P. Hamel. The ARG9 Gene Encodes the Plastid-Resident N-Acetyl Ornithine Aminotransferase in the Green Alga Chlamydomonas reinhardtii. Eukaryotic Cell (2009) 8: 1460-1463 Fields of Study Major Field: Plant Cellular and Molecular Biology v Table of Contents Abstract ............................................................................................................................... ii Vita ...................................................................................................................................... v List of Tables .................................................................................................................... xii List of Figures .................................................................................................................. xiii Chapter 1: Introduction ....................................................................................................... 1 1. Photosynthesis Overview ............................................................................................ 1 2. PSII Components......................................................................................................... 3 2.1 PSII Assembly ........................................................................................................... 5 2.2 D2 .............................................................................................................................. 7 2.2.1 HCF136 - Arabidopsis D2 Assembly Factor ......................................................... 8 2.2.2 YCF48 - Synechocystis ortholog of HCF136 ....................................................... 10 2.3 D1 ............................................................................................................................ 11 2.3.1 ALB3 - Arabidopsis Insertase .............................................................................. 12 2.3.2 ALB3.2 - Chlamydomonas ortholog of Arabidopsis ALB3 ................................ 14 2.3.3 LPA1 - Arabidopsis D1 Assembly Factor ........................................................... 17 vi 2.3.4 PAM68 - Arabidopsis PSII Assembly Factor ...................................................... 18 2.3.5 Psb27-H2 - Arabidopsis pD1 Assembly Factor ................................................... 20 2.3.6 HCF243 - Arabidopsis D1 Assembly Factor ....................................................... 21 2.4 CP43 ........................................................................................................................ 23 2.4.1 LPA2 and LPA3 - Arabidopsis CP43 Assembly Factors ..................................... 24 2.5 Assembly of the Manganese Cluster ....................................................................... 27 2.5.1 PratA - Synechocystis Mn Transporter and D1 Assembly Factor ........................ 28 2.5.2 Psb27 - Synechocystis Mn Cluster Assembly Factor ........................................... 30 2.6 PsbO ........................................................................................................................ 32 2.6.1 LTO1 - PsbO Assembly Factor ............................................................................ 32 2.6.2 Is there a PsbO reductase? .................................................................................... 33 2.7 PsbP ......................................................................................................................... 35 2.8 PsbP Related Proteins ............................................................................................. 37 2.8.1 Sll1418 - Synechocystis PsbP-like homologue..................................................... 37 2.9 PsbQ ........................................................................................................................ 38 2.9.1 Sll1638 - Synechocystis PsbQ-like homolog ........................................................ 39 3. b6f .............................................................................................................................. 40 3.1 Translation of petA .................................................................................................. 40 3.2 TCA1 - Chlamydomonas Translation Factor of petA ............................................. 41 vii 3.3 MCA1 - Post Transcriptional Factor of petA .......................................................... 42 3.4 Heme Attachment .................................................................................................... 48 3.4.1 CCS1 and CcsA - System II Heme Synthetase .................................................... 48 3.4.2 CcsBA - Bacterial Ortholog of CCS1 and CcsA ................................................. 49 3.4.3 ResB and ResC - B. subtilis orthologs of CCS1 and CcsA .................................. 50 3.4.4 What is the function of the b-type heme? ............................................................ 52 3.4.5 System II Variation .............................................................................................. 55 Chapter 2: CCS2, a novel factor required for cytochrome c assembly in the green alga Chlamydomonas reinhardtii ............................................................................................. 59 Abstract ......................................................................................................................... 59 Introduction ................................................................................................................... 60 RESULTS...................................................................................................................... 64 Cloning of CCS2 by functional complementation. ....................................................... 64 CCS2 encodes a 170 kDa protein of the OPR family. .................................................. 65 Localization. .................................................................................................................. 70 Essential domains are located in C-terminus. ............................................................... 71 Specific effects of the ccs2 mutation. ............................................................................ 71 MATERIALS AND METHODS .................................................................................. 72 Strains and culture condition. ........................................................................................ 72 Complementation. ......................................................................................................... 72 viii Constructs. ..................................................................................................................... 73 Immunoblots.................................................................................................................. 74 Chlorophyll Auto-fluorescence. .................................................................................... 74 GFP Fluorescence. ........................................................................................................ 75 OPR Protein Domains ................................................................................................... 75 DISCUSSION ............................................................................................................... 75 Identification of CCS2 as an OPR protein needed for cytochrome c assembly ............ 75 Unique Cytochrome