Mechanism of light regulation of Rubisco: a specific role for the larger Rubisco activase isoform involving reductive activation by thioredoxin-f

• Published in: Proceedings of the National Academy of Sciences - PNAS 1999-08, Vol.96 (16), p.9438-9443
• Main Authors: Zhang, N, Portis, A.R. Jr
• Format: Article
• Language: English
• Subjects: Adenosine Diphosphate - metabolism; Adenosine triphosphatases; adenosine triphosphate; Adenosine Triphosphate - metabolism; adenosinetriphosphatase; Amino Acid Sequence; Arabidopsis; Arabidopsis - enzymology; Arabidopsis - genetics; Arabidopsis thaliana; Base Sequence; Biological Sciences; Cells; Chloroplast Thioredoxins; Cloning, Molecular; deletions; Enzyme Activation; enzyme activity; enzyme polymorphism; Enzymes; Flowers & plants; Gene Expression Regulation, Enzymologic - radiation effects; Gene Expression Regulation, Plant - radiation effects; hydrolysis; Isoenzymes - genetics; Isoenzymes - metabolism; Kinetics; Light; Luminous intensity; Molecular Chaperones - genetics; Molecular Chaperones - metabolism; Molecular Sequence Data; mutants; Oxidation; Oxidation-Reduction; Physiological regulation; Plant Proteins - chemistry; Plant Proteins - genetics; Plant Proteins - metabolism; Plants; Polymerase chain reaction; Protein isoforms; Proteins; reduction; regulation; ribulose-bisphosphate carboxylase; Ribulose-Bisphosphate Carboxylase - metabolism; site-directed mutagenesis; Spinach; Spinacia oleracea; stimulation; sulfur proteins; Thioredoxin; Thioredoxins - metabolism
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.96.16.9438

Rubisco activase is a nuclear-encoded chloroplast protein that is required for the light activation of ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) in vivo. In most plants examined to date, there are two isoforms of Rubisco activase arising from alternative splicing that differ only at the carboxyl terminus. Here we demonstrate with recombinant proteins that in Arabidopsis the larger isoform has a unique role in the regulation of Rubisco activity. At physiological ratios of ADP/ATP, the 46-kDa isoform has minimal ATP hydrolysis and Rubisco activation activity in comparison with the 43-kDa isoform. Analysis of a series of carboxyl-terminal deletion and Ala substitution mutants of the 46-kDa isoform revealed that the presence of Cys residues at positions 411 and 392 were essential to preserve a low ATP hydrolysis and Rubisco activation activity in the presence of ADP. Consequently, incubation of the 46-kDa isoform with DTT and thioredoxin-f increased both activities, whereas incubations with DTT alone or with thioredoxin-m were ineffective. Thioredoxin-f and DTT had no effect on the 43-kDa isoform. However, premixing both isoforms before conducting a reduction and oxidation cycle demonstrated that the activity of both isoforms could be regulated. Reduction and oxidation also modulated the activity of native activase proteins isolated from either Arabidopsis or spinach, but not tobacco, which only has the smaller isoform. These findings suggest that in plants containing both isoforms, Rubisco activase regulates the activity of Rubisco in response to light-induced changes in both the ADP/ATP ratio and the redox potential via thioredoxin-f.