Effects of mutations at the two processing sites of the precursor for the small subunit of ribulose-bisphosphate carboxylase in Chlamydomonas reinhardtii

Abstract

The role of the two processing sites in the precursor of the small subunit (SS) of ribulose-1,5-bisphosphate carboxylase/oxygenase (pSS) of Chlamydomonas reinhardtii was studied by introducing mutations at the cleavage sites for the stromal processing peptidases SPP-1 and SPP-2, which hydrolyse wild-type pSS (20.6 kDa) to an intermediate-sized product iSS (18.3 kDa) and to the mature SS (16.3 kDa), respectively. The mutations introduced into cDNA resulted in exchange of (a) two amino acids flanking processing site 1, or (b) one or (c) both amino acids flanking processing site 2. Mutation (a) prevented pSS from being processed at site 1 but not from cleavage at site 2. Mutation (c) abolished the action of SPP-2 but not SPP-1. When pSS with mutation (c) was imported into isolated chloroplasts, iSS accumulated while SS formation was abolished. However, mature SS was produced even in the absence of iSS synthesis (mutation a). Import of pSS bearing mutation (b), which only partially inhibited processing at the SPP-2 site, slowed the rate of SS formation down whereas iSS and some slightly smaller derivatives accumulated. These experiments suggested that in Chlamydomonas processing of pSS can occur in two steps, whereby the first step is facultative. The same three mutations were studied in vivo after transformation of SS-deficient C. reinhardtii T60-3 with mutated genomic DNA. Growth and photosynthesis was as in control transformants, except for the slower-growing transformants (mutation c) where no mature SS was immuno-detected. However, pSS fragments with molecular masses between those of iSS and SS were present even in the ribulose-1,5-bisphosphate carboxylase/oxygenase holoenzyme.

Functional Hybrid Rubisco Enzymes with Plant Small Subunits and Algal Large Subunits

“…When Rubisco was purified from this strain and analyzed by SDS-PAGE, the small subunit was found to be about 2 kDa larger than the native spinach small subunit (data not shown). This observation was quite similar to that previously observed when small subunit transit peptide processing was blocked by directed mutant substitutions flanking the transit peptide-processing site (66). Because transit peptide processing in Chlamydomonas occurs in a two-step fashion, only 21 residues of the 45-residue transit peptide remained attached to the amino terminus of the mature small subunit sequence (66).…”

“…This observation was quite similar to that previously observed when small subunit transit peptide processing was blocked by directed mutant substitutions flanking the transit peptide-processing site (66). Because transit peptide processing in Chlamydomonas occurs in a two-step fashion, only 21 residues of the 45-residue transit peptide remained attached to the amino terminus of the mature small subunit sequence (66). Furthermore, transit peptide processing in Chlamydomonas may depend on residues next to those at the primary processing site (67).…”

“…3). Based on previous studies with the Chlamydomonas small subunit (66,67), it is likely that the nature of residue 2 plays a role in efficient transit peptide processing. In the x-ray crystal structures of Chlamydomonas and spinach Rubisco (63,64,70), residue 2 is in contact with and shields large subunit residues Trp-411, Pro-415, Pro-453, and Glu-454 from solvent, but there are no notable differences between the two structures in this region.…”

Modification of the Proteolytic Fragmentation Pattern upon Oxidation of Cysteines from Ribulose 1,5-Bisphosphate Carboxylase/Oxygenase

“…Although there have been several reports on proteolysis of Rubisco by exogenous ( , ), endogenous ( − ), or even chloroplastic ( − ) proteases, their activity has not been tested by comparing the effect on the reduced and oxidized carboxylase, with two exceptions. In one case, Rubisco was previously modified by ROS before being subjected to proteolysis ().…”