Alteration of haem-attachment and signal-cleavage sites for Paracoccus denitrificans cytochrome C550 probes pathway of c-type cytochrome biogenesis in Escherichia coli.
Paracoccus denitrificans cytochrome C550 is expressed as a periplasmic holo-protein in Escherichia coli; amino acid substitutions of cysteine residues in the haem-binding motif (Cys-X-X-Cys-His), either together or singly, prevented covalent attachment of haem but not polypeptide translocation into the periplasm. When the three alanine residues at positions -3 to -1 in the native signal-cleavage site were deleted, or alanine at -1 was changed to glutamine, signal cleavage was at alternative sites (after only ten residues in the latter case), but haem attachment still occurred. When the same three alanines were changed to Asp-Glu-Asp, a membrane-associated apo product that had retained the complete signal sequence was detected. These and other results presented here indicate that (i) haem attachment is not required for the apo-cytochrome C550 export to the periplasm; (ii) haem cannot attach to apo-cytochrome C550 when attached to the cytoplasmic membrane, suggesting that signal-sequence cleavage precedes periplasmic haem attachment, which can occur at as few as six residues from the mature N-terminus; and (iii) two cysteines are required for haem attachment, possibly because a disulphide bond is an intermediate. The gene for Saccharomyces cerevisiae mitochondrial iso-1-cytochrome c was expressed as a holo-protein in E. coli when fused with the signal sequence plus the first 10 residues of the mature cytochrome C550, indicating that the E. coli cellular apparatus for the c-type cytochrome biogenesis has a broad substrate specificity.
Sambongi
Y
Y
Department of Biochemistry, University of Oxford, UK.
Stoll
R
R
Ferguson
S J
SJ
D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D003574:Cytochrome c Group / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D017931:DNA Primers / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D015870:Gene Expression; D005798:Genes, Bacterial; D005800:Genes, Fungal; D006418:Heme / Q000378:metabolism; D015335:Molecular Probes; D008969:Molecular Sequence Data; D009154:Mutation; D010231:Paracoccus denitrificans / Q000235:genetics / Q000378:metabolism*; D021382:Protein Sorting Signals / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism
D003574:Cytochrome c Group; D017931:DNA Primers; D015335:Molecular Probes; D021382:Protein Sorting Signals; D006418:Heme; C031732:cytochrome C-550