Rubisco activase -- A typical AAA+ protein with unique features, and, PIFI -- A novel chloroplast protein functioning in chlororespiration

Rubisco activase facilitates the activation of Rubisco, which is powered by the ATP hydrolysis activity of activase and inhibited by ADP. In Arabidopsis, oxidation of the carboxyl-terminal (C) extension on the 46-kD isoform of activase significantly increases its ADP sensitivity for ATP hydrolysis and Rubisco activation. Replacing conserved negatively charged residues on the oxidized C-extension with alanine reduced the sensitivity of the mutants to ADP inhibition. The oxidized C-extension was crosslinked to the nucleotide-binding pocket of activase, with a higher efficiency in the presence of ADP or no nucleotides than ATP. Enzyme activities, nucleotide-binding and limited proteolysis assays clearly indicated that the C-extension interferes with not only ATP binding but also ATP hydrolysis, which appear to involve redox-mediated conformational changes of the C-extension. These results provide new details and insights into the mechanism of redox regulation of activase by its C-extension.; Two-conserved tryptophans, W109 and W250 in tobacco activase, were identified by site-directed mutagenesis as being responsible for the increased intrinsic fluorescence upon addition of ATP, which reports ATP-induced conformational changes. Substitution of W109 and F248 with other residues eliminated both enzyme activities and intrinsic fluorescence enhancement, without disturbing nucleotide-binding capacities. Substitution of F232 with Trp caused 40% decreases in enzyme activities but a 20% increase in intrinsic fluorescence upon binding ATP. These results provide support for the existence of a putative hydrophobic patch that is important for ATP-induced conformational changes.; A novel Arabidopsis chloroplast protein, named PIFI, with a similar C-extension as activase was identified as functioning in chlororespiration and possibly cyclic electron transport by reverse genetics. In a pifi mutant, which possesses an intact NDH complex, NDH-mediated dark reduction of plastoquinone was disrupted as indicated by its post-illumination fluorescence kinetics. The pifi mutant exhibited low capacity for thermal dissipation and a low oxidation level of the PSI reaction center, but similar electron transport efficiency of PSII as that of wild type, suggesting an impaired cyclic electron flow around PSI in the mutant. The PIFI protein is dispensable under standard growth conditions but important in protecting plants from photoinhibition and mild heat stress.