New insights into the role of CcmC, CcmD and CcmE in the haem delivery pathway during cytochrome c maturation by a complete mutational analysis of the conserved tryptophan-rich motif of CcmC.
Maturation of c-type cytochromes in Escherichia coli is a complex process requiring eight membrane proteins encoded by the ccmABCDEFGH operon. CcmE is a mediator of haem delivery. It binds haem transiently at a conserved histidine residue and releases it for directed transfer to apocytochrome c. CcmC, an integral membrane protein with six transmembrane helices, is necessary and sufficient to incorporate haem covalently into CcmE. CcmC contains a highly conserved tryptophan-rich motif, WGXXWXWD, in its second periplasmic loop. Here, we present the results of a systematic mutational analysis of this motif. Changes of the non-conserved T121 and W122 to A resulted in wild-type CcmC activity. Changes of the single amino acids W119A, G120A, W123A, W125I and D126A or of the spacing within the motif by deleting V124 (DeltaV124) inhibited the covalent haem incorporation into CcmE. Enhanced expression of ccmD suppressed this mutant phenotype by increasing the amounts of CcmC and CcmE polypeptides in the membrane. The DeltaV124 mutant showed the strongest defect of all single mutants. Mutants in which six residues of the tryptophan-rich motif were changed showed no residual CcmC activity. This phenotype was independent of the level of ccmD expression. Our results demonstrate the functional importance of the tryptophan-rich motif for haem transfer to CcmE. We propose that the three membrane proteins CcmC, CcmD and CcmE interact directly with each other, establishing a cytoplasm to periplasm haem delivery pathway for cytochrome c maturation.
Schulz
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Institut für Mikrobiologie, Eidgenössische Technische Hochschule, Schmelzbergstrasse 7, CH 8092 Zürich, Switzerland.
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Thöny-Meyer
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D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D001425:Bacterial Outer Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D001692:Biological Transport; D002462:Cell Membrane / Q000378:metabolism; D017124:Conserved Sequence; D003574:Cytochrome c Group / Q000378:metabolism*; D029968:Escherichia coli Proteins; D005816:Genetic Complementation Test; D006418:Heme / Q000378:metabolism*; D006420:Hemeproteins / Q000235:genetics / Q000378:metabolism*; D008565:Membrane Proteins / Q000235:genetics* / Q000378:metabolism*; D008969:Molecular Sequence Data; D009154:Mutation
D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins; C492058:CcmC protein, E coli; C116246:CcmC protein, bacteria; C415989:CcmD protein, E coli; C115384:CcmE protein, E coli; D003574:Cytochrome c Group; D029968:Escherichia coli Proteins; D006420:Hemeproteins; D008565:Membrane Proteins; D006418:Heme