Requirements for maturation of Bradyrhizobium japonicum cytochrome c550 in Escherichia coli.
Various forms of Bradyrhizobium japonicum cytochrome c550 (the cycA gene product) were overexpressed in Escherichia coli cells grown under different conditions. Antibodies directed against a synthetic cytochrome c550 peptide were used as tools to detect both, apoprotein and holoprotein. Complete maturation of the apoprotein into its holo form with haem covalently bound to the polypeptide was observed only under anaerobic growth conditions and in E. coli K12 derivatives, whereas haem binding did not occur in the E. coli BL21 host. When maturation was complete, holocytochrome c550 was found exclusively in the periplasmic fraction. A cycA-expressing plasmid construct lacking the genetic information for the signal sequence produced apoprotein that was rapidly degraded without further maturation. Mutations in the haem-binding site resulted in products that were translocated through the cytoplasmic membrane, but apparently became degraded. Our results support the view that attachment of haem to the apoprotein is not a prerequisite for cleavage of the signal sequence and occurs on the periplasmic side of the membrane, subsequent to translocation of the apoprotein precursor.
Thöny-Meyer
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L
Mikrobiologisches Institut, Eidgenössische Technische Hochschule, ETH-Zentrum, Zurich, Switzerland.
Künzler
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P
Hennecke
H
H
D000595:Amino Acid Sequence; D001483:Base Sequence; D001692:Biological Transport; D015153:Blotting, Western; D002462:Cell Membrane / Q000378:metabolism; D003574:Cytochrome c Group / Q000235:genetics* / Q000378:metabolism; D004269:DNA, Bacterial; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics / Q000254:growth & development; D006418:Heme / Q000378:metabolism; D008969:Molecular Sequence Data; D011499:Protein Processing, Post-Translational; D021382:Protein Sorting Signals / Q000235:genetics / Q000378:metabolism; D012231:Rhizobium / Q000201:enzymology*; D013347:Subcellular Fractions / Q000201:enzymology
D003574:Cytochrome c Group; D004269:DNA, Bacterial; D021382:Protein Sorting Signals; D006418:Heme; C031732:cytochrome C-550