A role for the disulfide bond spacer region of the Chlamydomonas reinhardtii coupling factor 1 gamma-subunit in redox regulation of ATP synthase.
The gamma-subunit of chloroplast coupling factor 1 contains a disulfide bond which is involved in the redox regulation of the enzyme. In all the sequence plant gamma-subunits this disulfide bond is separated by a five amino acid spacer region. To investigate the regulatory significance of this region genetic transformation experiments were performed with Chlamydomonas reinhardtii. C. reinhardtii strain atpC1 (nitl-305, cw 15, mt-), which does not accumulate the CF1 gamma-subunit polypeptide, was independently transformed with two constructs, each bearing mutations within the disulfide bond spacer region between Cys198 and Cys204 of the gamma-subunit. Successful complementation was confirmed by phenotypic selection, Northern blot analysis, and reverse transcription polymerase chain reaction. Whereas wild-type thylakoid membrane particles catalyze in vitro, PMS-dependent photophosphorylation that is stimulated 2-fold by the addition of DTT, similar particles from each of the mutant strains exhibit rates of ATP synthesis that are independent of DTT. Consistent with these results, wild-type CF1 ATPase activity is stimulated by DTT which is in contrast to the ATPase activities of both the mutant strains which are independent of DTT addition. These results suggest a role of the gamma-subunit disulfide bond spacer region in the redox regulation of chloroplast ATP synthase.
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
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D016825:Chlamydomonas reinhardtii / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D002736:Chloroplasts / Q000201:enzymology; D017931:DNA Primers / Q000235:genetics; D004220:Disulfides / Q000737:chemistry; D004229:Dithiothreitol / Q000494:pharmacology; D008773:Methylphenazonium Methosulfate / Q000494:pharmacology; D008969:Molecular Sequence Data; D009154:Mutation; D010084:Oxidation-Reduction; D010641:Phenotype; D010766:Phosphorylation; D010777:Photochemistry; D011487:Protein Conformation; D006180:Proton-Translocating ATPases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D014170:Transformation, Genetic
D017931:DNA Primers; D004220:Disulfides; D008773:Methylphenazonium Methosulfate; D006180:Proton-Translocating ATPases; D004229:Dithiothreitol