SUBSTRATE-SPECIFIC AND SPECIES-SPECIFIC PROCESSING ENZYMES FOR CHLOROPLAST PRECURSOR PROTEINS

Using different precursors of chloroplast proteins and stromal extract s from both Chlamydomonas reinhardii and pea chloroplasts, we analysed the specificity of stroma-localized processing peptidases. By gel fil tration of a stromal extract from isolated Chlamydomonas chloroplasts, fractions could be separated containing enzymic activities for proces sing the precursors of the small subunit of ribulose-1,5-bisphosphate carboxylase (pSS) and of the protein OEE1 from the photosynthetic wate r-splitting complex (pOEE1). The enzymes differed not only in molecula r size, but also in their sensitivity to inhibitors and in their pH op tima. Obviously, in the stroma of Chlamydomonas chloroplasts different peptidases exist for processing of pSS and pOEE1, the latter being co nverted into an intermediate-sized form, iOEE1, which was found to be further processed to mature OEE1 by a thylakoid-associated protease. T o study the species-specificity of the stromal peptidases, stromal ext racts from Chlamydomonas and pea chloroplasts were incubated with pSS from either of these organisms. In the heterologous combinations, the precursors were partly hydrolysed, but not to the correct size. In imp ortation assays, pSS from pea (but also the precursor of the ribosomal protein L12 from spinach) could not enter into chloroplasts from Chla mydomonas. In contrast, the algal pSS was imported into chloroplasts f rom pea, although it was not processed to mature SS. Our results indic ate that the importation machinery and the pSS-processing enzymes in h igher plants and green algae have different specificities and that in Chlamydomonas several stromal peptidases for different precursor prote ins exist.