Differential effects of N- and C-terminal deletions on the two activities of rubisco activase.

Spinach (Spinacea oleracea) leaf ribulose-1,5-bisphosphate carboxylase/oxygenase (rubisco) activase was subjected to limited proteolysis with trypsin and directed deletions were made by modifying the spinach rubisco activase cDNA and expressing the 41-kDa isoform in Escherichia coli. Protein exposed to trypsin displayed a more rapid loss of the ability to promote the activation of decarbamylated rubisco than ATP hydrolysis (e.g., 10 and 50% activity remaining, respectively, after 1 h). A series of N-terminal deletions exhibited near abolition of rubisco activation after the 12th residue, a conserved tryptophan, was deleted. Conversely, a deletion of 19 residues at the C-terminus increased rubisco activation with little effect on ATP hydrolysis, resulting in an increased efficiency of activation. The C-terminal deletion mutant was further modified by a site-directed mutation in the ATP binding region (Q109E) which was previously observed to increase the efficiency of activation (J. B. Shen and W. L. Ogren, 1991, Plant Physiol. 99, 1201-1207). The efficiency of activation with this double mutant was greater than that for either of the original mutants. The results indicate that a conserved tryptophan in the N-terminal portion of rubisco activase is critical for promotion of the activation of rubisco, consistent with a possible role in interaction with rubisco. The C-terminus appears to have a regulatory effect on both rubisco activation and ATP hydrolysis.