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.
Esau
B D
BD
Department of Plant Biology, University of Illinois, Urbana 61801, USA.
Snyder
G W
GW
Portis
A R
AR
D000595:Amino Acid Sequence; D018076:DNA, Complementary / Q000235:genetics; D004274:DNA, Recombinant / Q000235:genetics; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D017353:Gene Deletion; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010940:Plant Proteins; D012273:Ribulose-Bisphosphate Carboxylase / Q000235:genetics / Q000378:metabolism*; D016415:Sequence Alignment; D018724:Spinacia oleracea / Q000201:enzymology*
D018076:DNA, Complementary; D004274:DNA, Recombinant; D010940:Plant Proteins; C054765:rca protein, plant; D012273:Ribulose-Bisphosphate Carboxylase