Plant Molecular Biology 29: 53-61, 1995. © 1995 Kluwer Academic Publishers. Printed in Belgium. 53 Mutations in the processing site of the precursor of ribulose-l,5-bisphosphate carboxylase/oxygenase small subunit: effects on import, processing, assembly and stability Maggie Levy and Zach Adam* Department of Agricultural Botany, Faculty of Agriculture, The Hebrew University of Jerusalem, Rehovot 76100, Israel (* author for correspondence) Received 13 March 1995; accepted in revised form 8 June 1995 Key words: chloroplast, Pisum sativum, precursor, processing, Rubisco Abstract The small subunit (S SU) 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 pro- tein. To assess the importance of the Met residue for recognition and processing by the stromal pepti- dase, we substituted this residue with either Thr, Arg or Asp. The mutant precursor proteins were im- ported 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, co- migrating 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 mu- tant, 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 holoen- zyme 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. Introduction The small subunit (SSU) of ribulose-l,5-bispho- sphate carboxylase/oxygenase (Rubisco, EC 4.1.1.39), like most other chloroplast proteins, is encoded by nuclear genes, synthesized in the cy- tosol in a precursor form (pre-SSU), and imported post-translationally into the chloroplast. A cleavable N-terminal extension, designated the transit peptide, is necessary and sufficient to tar- get the protein to the chloroplast, mediate its re- cognition by a receptor component of the enve- lope, and translocate it into the organelle (for review, see [20]). During or after import, the pre- cursor is processed to its mature size by a stro- mal peptidase. Different functional regions can be distinguished within the transit peptide. The cen- tral region mediates binding of the precursor to the chloroplast envelope, whereas the N- and the C-terminal regions of the transit peptide are ira-