Mutations in the processing site of the precursor of ribulose-1,5-bisphosphate carboxylase/oxygenase small subunit: effects on import, processing, assembly and stability.
The small subunit (SSU) of Rubisco is synthesized in the cytosol in a precursor form. Upon import into the chloroplast, it is proteolytically processed at a Cys-Met bond to yield the mature form of the protein. To assess the importance of the Met residue for recognition and processing by the stromal peptidase, we substituted this residue with either Thr, Arg or Asp. The mutant precursor proteins were imported into isolated chloroplasts, and the products of the import reactions were analyzed. Mutants containing Thr or Arg residues at the putative processing site were processed to a single peptide, comigrating with the wild-type protein. N-terminal radio-sequencing revealed that these mutants were processed at the Cys-Thr and the Cys-Arg bond, respectively. After import of the Asp-containing mutant, four processed forms of the protein were observed. Analysis of the most abundant one, co-migrating with the wild-type protein, demonstrated that this species was also a product of correct processing, at the Cys-Asp bond. All the correctly processed peptides were found to be associated with the holoenzyme of Rubisco, and remained stable within the chloroplast, like the wild-type protein. The results of this study, together with previous ones, suggest that proper recognition and processing of the SSU precursor are more affected by residues N-terminal to the processing site than by the residue on the C-terminal side of this site.
Levy
M
M
Department of Agricultural Botany, Faculty of Agriculture, Hebrew University of Jerusalem, Rehovot, Israel.
Adam
Z
Z
D000595:Amino Acid Sequence; D001483:Base Sequence; D001692:Biological Transport; D004792:Enzyme Precursors / Q000235:genetics*; D004795:Enzyme Stability; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D011499:Protein Processing, Post-Translational / Q000235:genetics*; D012273:Ribulose-Bisphosphate Carboxylase / Q000235:genetics* / Q000378:metabolism
D004792:Enzyme Precursors; D012273:Ribulose-Bisphosphate Carboxylase