Volume 270,
Number 17,
Issue of April 28, pp. 9813-9818, 1995
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Role of the
Chlamydomonas reinhardtii Coupling Factor 1 
-Subunit
Cysteine Bridge in the Regulation of ATP Synthase
Stuart A.
Ross
,
Michael
X.
Zhang
,
Bruce R.
Selman
The 
-subunit of coupling factor 1 (CF
) 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 -subunit, was
transformed and complemented with -subunit constructs containing
amino acid substitutions localized to the cysteine bridge between
Cys
and Cys
. Successful complementation was
confirmed by phenotypic selection, Northern blot analysis, reverse
transcription polymerase chain reaction, and cDNA sequencing. CFATPase activities of the soluble enzymes were measured in the
presence and absence of dithiothreitol (DTT). Mutant CFenzymes 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 CFexhibits 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 -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.
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