Cytochrome c(6) from Cyanophora paradoxa - Characterization of the proteinand the cDNA of the precursor and import into isolated cyanelles

In the eukaryotic alga Cyanophora paradoxa, which does not contain plastocy anin, photosynthetic electron transport from the cytochrome b(6)/f complex to photosystem I is mediated by cytochrome c(6). Cytochrome c(6) was purifi ed to homogeneity by column chromatography and FPLC. The relative molecular mass of the holoprotein was determined by two different mass spectrometric methods (californium-252 plasma desorption and UV matrix-assisted laser de sorption ionization) giving 9251 +/- 3.3 Da. N-terminal Edman microsequenci ng yielded information on approx. 30 amino acid residues. Based on these da ta and on highly conserved regions of cytochromes c(6), degenerate oligonuc leotides were designed and used for PCR to amplify the genomic DNA of C. pa radoxa. Screening of a C. paradoxa cDNA library yielded several clones codi ng for preapo-cytochrome c(6). The deduced sequence of the mature protein w as verified by plasma desorption mass spectrometric peptide mapping and sho ws high similarity to those of cytochromes c(6) from cyanobacteria and alga e. Cytochrome c(6) appears to be encoded by a single nuclear gene (petJ) in C. paradoxa. As the mature protein is located in the lumen of the thylakoi d membrane, it has to traverse three biological membranes as well as the un ique peptidoglycan layer of the cyanelles before it reaches its final subce llular locale. Thus the transit sequence is composed of two different targe ting signals: a stroma targeting peptide resembling those of higher plants with respect to hydropathy plots and amino acid composition and a hydrophob ic signal peptide functioning as a thylakoid-traversing domain. There are i ndications for alternative sorting of part of the cyanelle cytochrome c(6) pool to the periplasmic space. This is the first known bipartite transit se quence of a cyanelle precursor protein from C. paradoxa, a model organism c oncerning the endosymbiotic origin of plastids. Labeled precursor is effici ently imported into isolated cyanelles, then routed into thylakoids and pro cessed to the mature protein. Hitherto, in vitro protein translocation was not reported for cyanobacterial-type thylakoids.