Thiol-disulfide exchange between nuclear-encoded and chloroplast-encoded subunits of pea acetyl-CoA carboxylase.
Fatty acid synthesis in pea chloroplasts is regulated by light/dark. The regulatory enzyme acetyl-CoA carboxylase is modulated by light/dark, presumably under redox regulation. Acetyl-CoA carboxylase is a multienzyme complex composed of biotin carboxylase and carboxyltransferase (CT). To demonstrate the redox regulation of CT, composed of the nuclear-encoded alpha and the chloroplast-encoded beta subunits, we identified the cysteine residues involved in such regulation. We expressed the recombinant CT in Escherichia coli and found that the partly deleted CT was, like the full-length CT, sensitive to a redox state. Site-directed mutagenesis of the deleted CT showed that replacement by alanine of the cysteine residue 267 in the alpha polypeptide or 442 in the beta polypeptide resulted in redox-insensitive CT and broke the intermolecular disulfide bond between the alpha and beta polypeptides. Similar results were confirmed in the full-length CT. These results indicate that the two cysteines in recombinant CT are involved in redox regulation by intermolecular disulfide-dithiol exchange between the alpha and beta subunits. Immunoblots of extract from plants incubated in the light or dark supported that such a disulfide-dithiol exchange is relevant in vivo. A covalent bond between a nuclear-encoded polypeptide and a chloroplast-encoded polypeptide probably regulates the enzyme activity in response to light.
Kozaki
A
A
Laboratory of Plant Molecular Biology, Graduate School of Agricultural Sciences, Nagoya University, Nagoya 464-8601, Japan.
Mayumi
K
K
Sasaki
Y
Y
D000103:Acetyl-CoA Carboxylase / Q000378:metabolism*; D000220:Adaptation, Biological; D000595:Amino Acid Sequence; D002467:Cell Nucleus / Q000235:genetics*; D002736:Chloroplasts / Q000201:enzymology* / Q000235:genetics*; D003545:Cysteine / Q000378:metabolism; D003553:Cystine / Q000378:metabolism; D004229:Dithiothreitol / Q000378:metabolism; D015971:Gene Expression Regulation, Enzymologic; D018506:Gene Expression Regulation, Plant; D008027:Light; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D018532:Pisum sativum / Q000201:enzymology* / Q000528:radiation effects; D021122:Protein Subunits; D017386:Sequence Homology, Amino Acid
D021122:Protein Subunits; D003553:Cystine; D000103:Acetyl-CoA Carboxylase; D003545:Cysteine; D004229:Dithiothreitol