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-bisphosphate carboxylase (pSS), which had been synthesized by using the cloned rbcS-2 gene of Chlamydomonas. SPP-1 shortened pSS to an intermediate-sized form (iSS), while SPP-2 cut pSS and iSS to the mature small subunit 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, apparent molecular masses of 340 kDa and 90 kDa were determined for SPP-1 and SPP-2, respectively. The comparison of these molecular masses with the protein patterns obtained by SDS/PAGE of the partially purified enzymes suggested that at least SPP-1 was a multimeric protein. The enzymes differed also in their pH optima of about 8 (SPP-1) and 9 (SPP-2) and in their sensitivity to different inhibitors. However, both enzymes 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 synthetic hexadecapeptide with 15 amino acids similar to the C-terminal end of the transit sequence of pSS, indicated that SPP-2 had some affinities not only to the transit sequence of pSS, but especially to sequences in the mature protein part. We conclude that SPP-2 in Chlamydomonas is the enzyme involved in import of pSS into chloroplasts and responsible for its processing by a one-step mechanism.
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Institute of Biochemistry, University of Bern, Switzerland.
Boschetti
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D000595:Amino Acid Sequence; D000818:Animals; D016825:Chlamydomonas reinhardtii / Q000201:enzymology* / Q000378:metabolism; D002736:Chloroplasts / Q000378:metabolism*; D010450:Endopeptidases / Q000378:metabolism*; D006868:Hydrolysis; D008969:Molecular Sequence Data; D010940:Plant Proteins / Q000378:metabolism*; D011498:Protein Precursors / Q000737:chemistry / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D017154:Stromal Cells / Q000201:enzymology; D013379:Substrate Specificity
D010940:Plant Proteins; D011498:Protein Precursors; D010450:Endopeptidases