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 proteolyticall y 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 proce ssing by the stromal peptidase, we substituted this residue with eithe r Thr, Arg or Asp. The mutant precursor proteins were imported into is olated chloroplasts, and the products of the import reactions were ana lyzed. Mutants containing Thr or Arg residues at the putative processi ng 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. Afte r import of the Asp-containing mutant, four processed forms of the pro tein were ob served. Analysis of the most abundant one, co-migrating w ith the wild-type protein, demonstrated that this species was also a p roduct of correct processing, at the Cys-Asp bond. All the correctly p rocessed peptides were found to be associated with the holoenzyme of R ubisco, and remained stable within the chloroplast, like the wild-type protein. The results of this study, together with previous ones, sugg est that proper recognition and processing of the SSU precursor are mo re affected by residues N-terminal to the processing site than by the residue on the C-terminal side of this site.