A processing intermediate of a stromal chloroplast import protein in Chlamydomonas

Proteins synthesized in the cytoplasm and destined for importation into the chloroplast across the double envelope membrane contain an N-terminal tran sit sequence which upon import is cleaved off by a stromal-processing pepti dase. Since for stromal-residing proteins no intermediates have ever been f ound in vivo, it is assumed that precursor proteins are cleaved to the matu re size by one proteolytic event which occurs immediately after translocati on across both envelope membranes. During import of the precursor of the sm all subunit of ribulose-1,5-bisphosphate carboxylase (pSS) into isolated ch loroplasts of Chlamydomonas we identified an intermediate-sized product, ca lled iSS, It might be identical to a previously described ISS obtained in v itro 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 subun it. (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 th e envelope, as suggested by protease treatment of chloroplasts, questions t he one-step translocation mechanism of precursor import into chloroplasts.