PARTIAL-PURIFICATION AND PROPERTIES OF ENZYMES INVOLVED IN THE PROCESSING OF A CHLOROPLAST IMPORT PROTEIN FROM CHLAMYDOMONAS-REINHARDII

Two stromal peptidases (SPP-1 and SPP-2) were partially purified from chloroplasts of Chlamydomonas reinhardii. They specifically processed in vitro the precursor of the small subunit of ribulose-1, 5-bisphosph ate carboxylase (pSS), which had been synthesized by using the cloned rbcS-2 gene of Chlamydomonas. SPP-1 shortened pSS to an intermediate-s ized form (iSS), while SPP-2 cut pSS and iSS to the mature small subun it SS. N-terminal amino acid sequencing demonstrated that the reaction product obtained with SPP-2 had an N-terminus identical to natural SS , and that iSS derived from pSS by hydrolysis at the amino side of the methionine located within the transit sequence. By gel filtration, ap parent molecular masses of 340 kDa and 90 kDa were determined for SPP- 1 and SPP-2, respectively. The comparison of these molecular masses wi th the protein patterns obtained by SDS/PAGE of the partially purified enzymes suggested that at least SPP-1 was a multimeric protein. The e nzymes differed also in their pH optima of about 8 (SPP-1) and 9 (SPP- 2) and in their sensitivity to different inhibitors. However, both enz ymes seem to be serine proteases as they were completely blocked by N- alpha-tosyl-L-lysinechloromethane or tosylphenylalaninechloromethane, respectively. Competition experiments, using either mature SS or a syn thetic hexadecapeptide with 15 amino acids similar to the C-terminal e nd of the transit sequence of pSS, indicated that SPP-2 had some affin ities not only to the transit sequence of pSS, but especially to seque nces in the mature protein part. We conclude that SPP-2 in Chlamydomon as is the enzyme involved in import of pSS into chloroplasts and respo nsible for its processing by a one-step mechanism.