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.

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].…”

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

“…By fractionation of such chloroplasts into supernatant and membranes, SS and iSS were found to be soluble proteins while pSS remained bound to the membrane fraction. Furthermore, treatment of chloroplasts after pSS import both with thermolysin, which degrades proteins outside the outer envelope membrane, and with trypsin, acting additionally in the intermembrane space, revealed that iSS but not SS was located in the intermembrane space of the chloroplast envelope [14]. These findings raised the question about the physiological role of iSS and the enzyme SPP-1.…”

“…In itro, a stromal protein fraction enriched in SPP-2 cleaved pSS (20.6 kDa) at the N-terminal side of Met-46 (processing site 2), thereby producing the ' mature ' protein small subunit (SS ; 16.3 kDa), whereas SPP-1 cleaved pSS N-terminally of Met-25 in the middle of the pre-sequence (processing site 1), producing the intermediate-sized form iSS (18.3 kDa) [13]. iSS could also be detected after import of heavily labelled pSS into isolated chloroplasts [14]. By fractionation of such chloroplasts into supernatant and membranes, SS and iSS were found to be soluble proteins while pSS remained bound to the membrane fraction.…”

Evolution of Protein Targeting into “Complex” Plastids: The “Secretory Transport Hypothesis”

“…were subsequently modified by coevolution with the respective receptor associated with that translocon. This might explain the modular structure of transit peptides in plants (Bruce, 2000) and previous results indicating that the N-terminal part of the SSU protein leader peptide in Chlamydomonas is processed to an intermediate form in the envelope intermembrane space upon import into chloroplasts (Su et al, 1999).…”