Assembly of cytochrome f into the cytochrome bf complex in isolated pea chloroplasts.
Structural features of cytochrome f necessary for assembly into the cytochrome bf complex were examined in isolated pea chloroplasts following import of (35)S-labelled chimeric precursor proteins, consisting of the presequence of the small subunit of Rubisco fused to the turnip cytochrome f precursor. Assembly was detected by nondenaturing gel electrophoresis of dodecyl maltoside-solubilized thylakoid membranes. A cytochrome f polypeptide unable to bind haem because of mutagenesis of Cys21 and Cys24 to alanine residues was assembled into the complex and had similar stability to the wild-type polypeptide. This indicates that covalent haem binding to cytochrome f is not necessary for assembly of the protein into the cytochrome bf complex. A truncated protein lacking the C-terminal 33 amino acid residues, including the transmembrane span and the stroma-exposed region, was translocated across the thylakoid membrane, had a similar stability to wild-type cytochrome f but was not assembled into the complex. This indicates that the C-terminal region of cytochrome f is important for assembly into the complex. A mutant cytochrome f unable to bind haem and lacking the C-terminal region was also translocated across the thylakoid membrane but was extremely labile, indicating that, in the absence of the C-terminal membrane anchor, haem-less cytochrome f is recognized by a thylakoid proteolytic system.
Mould
R M
RM
Department of Plant Sciences, University of Cambridge, UK. Ruth.Mould@plantsci.cam.ac.uk
Kapazoglou
A
A
Gray
J C
JC
D000409:Alanine / Q000737:chemistry; D000596:Amino Acids / Q000737:chemistry; D001937:Brassica / Q000378:metabolism; D002736:Chloroplasts / Q000378:metabolism*; D003545:Cysteine / Q000737:chemistry; D003573:Cytochrome b Group / Q000737:chemistry* / Q000378:metabolism*; D045346:Cytochrome b6f Complex; D003580:Cytochromes / Q000737:chemistry* / Q000378:metabolism*; D045348:Cytochromes f; D018076:DNA, Complementary / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D018532:Pisum sativum / Q000378:metabolism*; D011485:Protein Binding; D014176:Protein Biosynthesis; D011498:Protein Precursors / Q000378:metabolism; D012273:Ribulose-Bisphosphate Carboxylase / Q000378:metabolism; D013347:Subcellular Fractions; D020524:Thylakoids / Q000378:metabolism; D013997:Time Factors; D014158:Transcription, Genetic
D000596:Amino Acids; D003573:Cytochrome b Group; D003580:Cytochromes; D018076:DNA, Complementary; D011498:Protein Precursors; D045346:Cytochrome b6f Complex; D045348:Cytochromes f; D012273:Ribulose-Bisphosphate Carboxylase; D003545:Cysteine; D000409:Alanine