UBC Theses and Dissertations
Roles of the proteins PuhB, PuhC, PuhE, PufQ, and PufX in photosynthesis by Rhodobacter capsulatus Aklujkar, Muktak Ashok
The photosynthetic apparatus of anoxygenic purple bacteria such as Rhodobacter capsulatus is a remarkable example of membrane protein organization. Much remains to be understood about the factors that govern the proportionate synthesis of pigments and proteins, the assembly and maintenance of pigment-protein complexes in the membrane, and the long-range organization of these complexes through protein-protein interactions. The puh operon of R. capsulatus contains four genes, the first encoding a polypeptide of the photosynthetic reaction centre (RC). The remaining genes: puhB, puhC, and puhE, are found in all purple phototrophic bacteria examined to date. This study examines the roles of the three proteins PuhB, PuhC, and PuhE in assembly and decay of the RC and of the associated antenna called light harvesting complex 1 (LH1), in phototrophic growth, and in interactions with other proteins. Serendipitously, each protein was found to have a different functional relationship to PufQ, a protein implicated in many aspects of photosynthetic apparatus biogenesis, and new roles were discovered for PufX, a polypeptide of the RC-LH1 complex. Overall, the results emphasize the interrelatedness of assembly processes of the RC and LH1. . All three predicted transmembrane (TM) segments of PuhB were found to span a bacterial inner membrane, and the second TM segment was capable of self-association. In the absence of PuhB, the amount of RC was as little as 12% of the wild type level, and it did not bind bacteriochlorophyll (BChl) properly. There was an RC-dependent near-total loss of LH1, the PufX protein was almost completely absent, and cells required at least 12 hours to adapt to anaerobic phototrophic growth. A tag at the N-terminus of PuhB prevented complementation in trans, but co-translation with PufQ eliminated the lag and restored the specific growth rate to 84% of wild type. A plausible model for the function of PuhB is that a PuhB dimer co-operates with PufQ to assist in RC assembly. Without PuhC in the membrane, phototrophic growth was sustained with difficulty, and benefited from 48 hours, compared to 24 hours, of semiaerobic pre-incubation. PuhC of R. capsulatus could be substituted perfectly with PuhC of Rhodobacter sphaeroides, but distantly related PuhC proteins improved the specific growth rate from 14% of wild type to between 19% and 24%. The puhC growth defect depended on pufQ and puhE, genes that regulate BChl biosynthesis, and was mitigated by downregulation or loss of light harvesting complex 2 (LH2). When PufX was transcribed separately from the RC-LH1 proteins, its level was reduced when puhC was deleted. Because PuhC was required for optimum levels of the RC-LH1 complex, but proved to be at most a minor determinant of RC assembly and LH1 assembly, the role of PuhC may be to expand and reorganize the RC-LH1 core complex as a whole. A puhE deletion created a minor obstacle to the transition from aerobic respiratory growth to active phototrophy that could not be complemented in trans. PuhE, an integral membrane protein with seven predicted TM segments, was found to inhibit BChl production by 49%, counterbalancing PufQ, and reduced the individual rates of RC and LH1 assembly by about 30% and 26%, respectively, without affecting expression of the polypeptides. However, the puhE deletion reduced the steady-state level of RC-LH1 by as much as 53% under high light intensity. Therefore, PuhE may perform two functions: to modulate BChl biosynthesis and/or degradation in response to light intensity; and to direct BChl into RC-specific and LHl-specific pathways. When the puf operon encoding the RC and LH1 (as well as PufX) was deleted and portions of the puf operon were restored on a plasmid, the final amount of RC-specific absorption was increased by 74% by leaving the chromosomal pufX gene intact, only in the presence of PufB, the outer polypeptide of LH1, and only in the absence of the inner LH1 polypeptide, Puf A. PufX was not immunodetected at stoichiometric levels in the absence of any RC-LH1 polypeptide or PuhB. The pufX merodiploid strains expressed more PufX protein than strains that contained a single pufX gene, and the extra protein reduced the level of LH1 by 14% only in the presence of the RC. The chromosomal pufii. gene produced more protein but it affected LH1 absorption less (25% reduction) than PufX produced from the co-transcribed gene (43% reduction). The single T M segments of PufX from both R. capsulatus and R. sphaeroides were capable of homodimerization. Therefore, PufX could be the axis of twofold symmetry in dimers of RC-LH1, and may form an oligomer when overexpressed, by interacting with PufB, to enhance RC assembly. In short, this work has furthered our understanding of five important photosynthesis proteins.
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