Mutations at the transit peptide-mature protein junction separate two cleavage events during chloroplast import of the chlorophyll a/b-binding protein.

摘要: Abstract We have shown previously that during in vitro import into chloroplasts, the precursor of major light-harvesting chlorphyll a/b-binding protein (LHCP) generated from a wheat gene gives rise to two mature forms (25 and approximately 26 kDa) which are inserted thylakoids. However, incubation LHCP with chloroplast-soluble extract an organelle-free processing reaction, NH2 terminus is cleaved, yielding only 25-kDa peptide. In present study, mutations at transit peptide-mature junction were introduced investigate relationship between peptides determinants proteolytic processing. Mutant p delta 3 lacks amino acids including Met34 primary cleavage site thought give 26-kDa It still processed reaction both assays + 4 has immediately after proline disrupts alpha-helix predicted by Garnier-Osguthorpe-Robson method (Garnier, J., Osguthorpe, D. Robson, B. (1978) J. Mol. Biol. 120, 97-120) extend through this region. Although imported, it inefficiently processed; 25- peptide found, but least 60% imported remains uncleaved. Less than 5% assay. Replacement mutant alpha restores upon chloroplast, mutant, also 4-amino acid insert, yields not reaction. These results provide evidence obtained result independent sites: first Met34, second 10 downstream within what been designated protein. Whereas removed retains functional site, or one very near it, accessible enzyme import. The conditions specific for secondary site. discuss implications these findings terms heterogeneity vivo.