Essential histidine and tryptophan residues in CcsA, a system II polytopic cytochrome c biogenesis protein.
Three distinct systems (I, II, and III) for catalysis of heme attachment to c-type apocytochromes are known. The CcsA and Ccs1 proteins are required in system II for the assembly of bacterial and plastid cytochromes c. A tryptophan-rich signature motif (WWD), also occurring in CcmC and CcmF found in system I, and three histidinyl residues, all strictly conserved in CcsA suggest a function in heme handling. Topological analysis of plastid CcsA in bacteria using the PhoA and LacZalpha reporters placed the WWD motif, the conserved residues His(212) and His(347) on the lumen side of the membrane, whereas His(309) was assigned a location on the stromal side. Functional analysis of CcsA through site-directed mutagenesis enabled the designation of the initiation codon of the ccsA gene and established the functional importance of the WWD signature motif and the absolute requirement of all three histidines for the assembly of plastid c-type cytochromes. In a ccsA mutant, a 200-kDa Ccs1-containing complex is absent from solubilized thylakoid membranes, suggesting that CcsA operates together with Ccs1. We propose a model where the WWD motif and histidine residues function in relaying heme from stroma to lumen and we postulate the existence of a cytochrome c assembly machinery containing CcsA, Ccs1 and additional components.
Hamel
Patrice P
PP
Department of Chemistry and Biochemistry, UCLA, Los Angeles, California 90095-1569, USA.
Dreyfuss
Beth Welty
BW
Xie
Zhiyi
Z
Gabilly
Stéphane T
ST
Merchant
Sabeeha
S
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D001692:Biological Transport; D002696:Chlamydomonas; D002736:Chloroplasts / Q000378:metabolism; D017124:Conserved Sequence; D003574:Cytochrome c Group / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D006418:Heme / Q000737:chemistry; D006639:Histidine / Q000737:chemistry*; D008954:Models, Biological; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D009687:Nuclear Proteins / Q000378:metabolism*; D010641:Phenotype; D015800:Protozoan Proteins; D011993:Recombinant Fusion Proteins; D013997:Time Factors; D014364:Tryptophan / Q000737:chemistry*
D003574:Cytochrome c Group; D009687:Nuclear Proteins; D015800:Protozoan Proteins; D011993:Recombinant Fusion Proteins; C075103:ccsA protein, protozoan; D006418:Heme; D006639:Histidine; D014364:Tryptophan