Isolation and characterization of a Chlamydomonas reinhardtii mutant lacking the gamma-subunit of chloroplast coupling factor 1 (CF1).

Abstract

Several nonphotoautotrophic mutants of Chlamydomonas reinhardtii were generated by transforming strain nitl-305 (cw 15) with exogenous DNA. An enrichment for potential photophosphorylation mutants was performed on medium containing arsenate, and acetate-requiring auxotrophs were then identified by replica plating. Strains containing a potential mutation in the nuclear DNA encoding the chloroplast coupling factor 1 (CFI) -y-subunit (the atpC gene) were first identified serologically with a monospecific antiserum directed against the CF1 fy-subunit polypeptide. Of several mutants isolated, one, designated T1-54, was characterized at the protein, DNA, and RNA levels. Mutant strain T1-54 lacks anti-CF1 y-subunit cross-reacting material, exhibits polymorphism at the atpC locus compared with the parental strain, and lacks the mRNA transcript for the CFI y-subunit. The data are consistent with there being an insertion of exogenous DNA, a deletion of DNA, or both at the 5' end of the gene encoding the CF1 y-subunit.The proton-translocating ATP synthase (chloroplast coupling factor CFO-CF,) complex in chloroplast thylakoid membranes couples the chemical potential energy in the proton motive force to the synthesis of ATP. The complex consists of two discrete sectors, CFo and CF1. CFo is an intrinsic thylakoid membrane complex, contains four different types of polypeptides (designated I through IV), and functions as a proton translocase. CF1 is an extrinsic membrane complex, contains five different types of polypeptides (a through E), and has the catalytic sites for ATP synthesis and hydrolysis (15).The molecular mechanism of CF1-catalyzed ATP synthesis is not well understood. However, ATP synthases from a variety of organisms appear to have similar mechanisms. Consistent with this is the observation that the subunit stoichiometry (a3P3Y8E) of CF1 is rigorously conserved and the amino acid sequences of these subunits are highly conserved, especially those involved in catalysis (a, ,B, and -y) (15). Unlike the reaction mechanism, the regulation of ATP synthase activity depends on both the species and subcellular organelle with which they are associated (1). One unique feature of the chloroplast complex is that its catalytic activity is regulated, in part, by the oxidation state of the y-subunit (9,17).Although there is some diversity in the amino acid composition of the -y-subunit among different plant species, one common feature seems to be a pair of cysteine residues that form an intrapeptide disulfide bridge (18). In the absence of a proton motive force, under conditions in which the catalytic activity of the ATP synthase is latent, the y-subunit cystine bridge is inaccessible to hydrophilic thiol reagents. The development of a proton motive force across the thylakoid membrane not only provides the driving force for ATP synthesis but also causes substantial conformational changes in the coupling factor (18). These conformational changes result in the activation of ATP synthase activity and the * Corresponding author. exp...

Inactivation of the Chloroplast ATP Synthase γ Subunit Results in High Non-photochemical Fluorescence Quenching and Altered Nuclear Gene Expression in Arabidopsis thaliana

“…Biogenesis of ATP synthases in eubacteria and mitochondria proceeds through assembly of the soluble F 1 followed by association to the transmembrane F 0 subcomplex (17,60). The independence of assembly of the two subcomplexes is abolished in plastids of the green algae Chlamydomonas where a concerted assembly of CF 1 and CF 0 subunits has been reported for mutants affected in different subunits (61)(62)(63). The same holds true for dpa1, where lack of the nuclear encoded ␥ subunit affects the accumulation of all other nuclear and plastid-encoded subunits that constitute the ATP synthase.…”

The Regulation of Carbonic Anhydrase and Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase Activase by Light and CO2 in Chlamydomonas reinhardtii

“…RNA was extracted from C. reinhardtii cultures using the method of Smart and Selman (1991). Northern blots were performed essentially as described by Sambrook et al (1989), using a full-length Rubisco activase cDNA clone (kindly provided by Dr. A. Portis, University of Illinois, Urbana), and a partial Cahl cDNA clone (kindly provided by Dr. M. Spalding, Iowa State University, Ames).…”

Thioredoxins: structure and function in plant cells

“…Very elegant experiments have, however, been conducted taking advantage of the isolation of a C reinhardtii mutant lacking the y subunit of chloroplast coupling factor 1 (Smart & Selman, 1991). Mutated versions of the y subunit have in turn been reintroduced, confirming the role of this nine aminoacid insertion, as well as of the disulphide bond spacer region (Smart & Selman, 1993;Ross, Zhang & Selman, 1995, 1996.…”