Conversion of cytochrome f to a soluble form in vivo in Chlamydomonas reinhardtii.
We introduced a stop codon in place of the ATT codon encoding Ile283 (numbered from the Met initiation codon) in the petA gene from Chlamydomonas reinhardtii. The resulting protein was expected to be truncated on its carboxy-terminus end, lacking the last 35 amino acids. This region of the polypeptide sequence encompasses a hydrophobic stretch assumed to anchor the protein in the thylakoid membrane. Once introduced in whole cells of C. reinhardtii by chloroplast transformation, the modified petA gene expressed a truncated apoprotein which was efficiently converted to a truncated holocytochrome f. This protein accumulated in the lumen of the thylakoids in a soluble form. Thus the conversion of preapocytochrome f to holocytochrome f does not require an interaction with the membrane through its C-terminus anchor. We show that the rest of the cytochrome b6f complex failed to accumulate in the transformants, most probably because of a lack of interaction between soluble cytochrome f and the other cytochrome b6f subunits. However, soluble cytochrome f was still able to donate electrons to photosystem I, which is indicative of its ability to maintain interactions with plastocyanin. The control of the rate of synthesis of cytochrome f by the neighboring subunit, suIV (Kuras & Wollman (1994) EMBO J. 13, 1019-1027), was not observed with the truncated cytochrome f. This observation suggests that either the transmembrane anchor of cytochrome f contains a target for the regulation of cytochrome f translation by suIV or there is a transient form of membrane-bound cytochrome f which is highly sensitive to proteolysis at an early post-translational stage.
Kuras
R
R
Service de Photosynthèse, Institut de Biologie Physico-Chimique, Paris, France.
Wollman
F A
FA
Joliot
P
P
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D016825:Chlamydomonas reinhardtii / Q000235:genetics / Q000378:metabolism*; D003062:Codon; D003580:Cytochromes / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D045348:Cytochromes f; D017931:DNA Primers; D017343:Genes, Plant; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism
D003062:Codon; D003580:Cytochromes; D017931:DNA Primers; D011994:Recombinant Proteins; D045348:Cytochromes f