A processing intermediate of a stromal chloroplast import protein in Chlamydomonas

Abstract

Proteins synthesized in the cytoplasm and destined for importation into the chloroplast across the double envelope membrane contain an N-terminal transit sequence which upon import is cleaved off by a stromal-processing peptidase. Since for stromal-residing proteins no intermediates have ever been found in vivo, it is assumed that precursor proteins are cleaved to the mature size by one proteolytic event which occurs immediately after translocation across both envelope membranes. During import of the precursor of the small subunit of ribulose-1,5-bisphosphate carboxylase (pSS) into isolated chloroplasts of Chlamydomonas we identified an intermediate-sized product, called iSS. It might be identical to a previously described iSS obtained in vitro by a partially purified soluble chloroplast protease [Su and Boschetti (1993) Eur. J. Biochem. 217, 1039-1047]. The kinetics of the formation of iSS in chloroplasts suggest that pSS is processed to the mature small subunit (SS) not by one, but by two steps via this intermediate product. Since, after an induction period, the ratio of iSS/SS was constant under various experimental conditions of import, the formation of iSS was considered not to be a side-reaction. The location of iSS in the intermembrane space of the envelope, as suggested by protease treatment of chloroplasts, questions the one-step translocation mechanism of precursor import into chloroplasts.

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

“…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). Taking these observations into account, we wondered whether the nature of residue 2 in the mature small subunit sequence might influence transit peptide processing.…”

“…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.…”

Varying competence for protein import into chloroplasts during the cell cycle in Chlamydomonas

“…Both proteins were thermolysin resistant and therefore located within the chloroplast. iSS had been localized to the intermembrane space of the envelope [32].…”