Role of the Chlamydomonas reinhardtii coupling factor 1 gamma-subunit cysteine bridge in the regulation of ATP synthase.
The gamma-subunit of coupling factor 1 (CF1) contains a cysteine bridge that is thought to be involved in the redox control of enzymatic activity. In order to test the regulatory significance of this disulfide bond, genetic transformation experiments with Chlamydomonas reinhardtii were performed. C. reinhardtii strain atpC1 (nit1-305, cw 15, mt-), which is null for the gamma-subunit, was transformed and complemented with gamma-subunit constructs containing amino acid substitutions localized to the cysteine bridge between Cys198 and Cys204. Successful complementation was confirmed by phenotypic selection, Northern blot analysis, reverse transcription polymerase chain reaction, and cDNA sequencing. CF1 ATPase activities of the soluble enzymes were measured in the presence and absence of dithiothreitol (DTT). Mutant CF1 enzymes showed no effect of DTT although increased activity was observed for the wild-type enzyme. In vitro, phenazine methosulfate-dependent photophosphorylation assays revealed that wild-type CF1 exhibits a 2-fold stimulation in the presence of 25 mM DTT, whereas each of the mutant enzymes has activities that are DTT-independent. Growth measurements indicated that despite the absence of a regulatory disulfide/dithiol, the mutant strains grew with the same kinetics as wild type. This study provides evidence to illustrate the involvement of the gamma-subunit in the redox regulation of ATP synthesis in vivo. This work is also the first demonstration in C. reinhardtii of stable nuclear transformation using mutated genes to complement a known defect.
Ross
S A
SA
Department of Biochemistry, College of Agricultural and Life Sciences, University of Wisconsin, Madison 53706, USA.
Zhang
M X
MX
Selman
B R
BR
D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D016825:Chlamydomonas reinhardtii / Q000201:enzymology*; D003001:Cloning, Molecular; D003545:Cysteine / Q000378:metabolism*; D017931:DNA Primers; D018076:DNA, Complementary; D008773:Methylphenazonium Methosulfate / Q000494:pharmacology; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D050901:Nitrate Reductase; D009565:Nitrate Reductases / Q000235:genetics; D010084:Oxidation-Reduction; D010766:Phosphorylation; D016133:Polymerase Chain Reaction; D006180:Proton-Translocating ATPases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D014170:Transformation, Genetic
D017931:DNA Primers; D018076:DNA, Complementary; D012333:RNA, Messenger; D008773:Methylphenazonium Methosulfate; D009565:Nitrate Reductases; D050901:Nitrate Reductase; D006180:Proton-Translocating ATPases; D003545:Cysteine