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 re gulatory enzyme acetyl-CoA carboxylase is modulated by light/dark, presumab ly 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 a and the chlo roplast-encoded beta subunits, we identified the cysteine residues involved in such regulation. We expressed the recombinant CT in Escherichia coli an d 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 r eplacement by alanine of the cysteine residue 267 in the a polypeptide or 4 42 in the beta polypeptide resulted in redox-insensitive CT and broke the i ntermolecular disulfide bond between the a and beta polypeptides. Similar r esults were confirmed in the full-length CT. These results indicate that th e two cysteines in recombinant CT are involved in redox regulation by inter molecular disulfide-dithiol exchange between the alpha and beta subunits. I mmunoblots 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 polypeptid e probably regulates the enzyme activity in response to light.