Chimeric fusions of subunit IV and PetL in the b6f complex of Chlamydomonas reinhardtii: structural implications and consequences on state transitions.
The cytochrome b(6)f complex of Chlamydomonas reinhardtii contains four large subunits and at least three small ones, PetG, PetL, and PetM, whose role and location are unknown. Chimeric proteins have been constructed, in which the C terminus of subunit IV is fused to either one or the other of the two putative N termini of PetL. Biochemical and functional analysis of the chimeras together with mass spectrometry analysis of the wild-type (WT) complex led to the following conclusions: (i) neither a free subunit IV C terminus nor a free PetL N terminus is required for assembly of the b(6)f complex; (ii) the first AUG codon in the sequence of the gene petL is used for initiation; (iii) the N terminus of WT PetL lies in the lumen; (iv) in the WT complex, the N terminus of PetL and the C terminus of subunit IV are within reach of each other; (v) the purified b(6)f complex from C. reinhardtii contains an eighth, hitherto unrecognized subunit, PetN; and (vi) the ability to perform state transitions is lost in the chimeric mutants, although (vii) the Q-cycle is unaffected. A structural hypothesis is presented to account for this peculiar phenotype.
Zito
Francesca
F
UMR 7099, CNRS and Université Paris-7, Institut de Biologie Physico-Chimique, 13 rue Pierre et Marie Curie, Paris F-75005, France. francesca.zito@ibpc.fr
Vinh
Joelle
J
Popot
Jean-Luc
JL
Finazzi
Giovanni
G
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D016825:Chlamydomonas reinhardtii / Q000378:metabolism*; D003062:Codon; D003573:Cytochrome b Group / Q000737:chemistry*; D045346:Cytochrome b6f Complex; D015151:Immunoblotting; D013058:Mass Spectrometry; D008954:Models, Biological; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000737:chemistry* / Q000378:metabolism; D010084:Oxidation-Reduction; D010641:Phenotype; D010766:Phosphorylation; D010940:Plant Proteins; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D015800:Protozoan Proteins / Q000737:chemistry; D011993:Recombinant Fusion Proteins / Q000737:chemistry* / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013050:Spectrometry, Fluorescence; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D020524:Thylakoids / Q000378:metabolism; D013997:Time Factors
D003062:Codon; D003573:Cytochrome b Group; D009687:Nuclear Proteins; C098901:PetM protein, Chlamydomonas reinhardtii; D010940:Plant Proteins; D015800:Protozoan Proteins; D011993:Recombinant Fusion Proteins; C075416:petG protein, Chlamydomonas reinhardtii; D045346:Cytochrome b6f Complex