THE PEA CHLOROPLAST MEMBRANE-ASSOCIATED PROTEIN, IEP96, IS A SUBUNIT OF ACETYL-COA CARBOXYLASE

Two forms of acetyl-CoA carboxylase (ACCase) have been characterized i n pea (Pisum sativum L.) leaves; a heteromeric chloroplast enzyme and a homomeric, presumably cytosolic enzyme. The biotin carboxylase (BC), biotin carboxyl carrier protein (BCCP), and beta-carboxyltransferase (CT) subunits of the plastidial-ACCase have recently been characterize d and cloned. To further characterize the carboxyl-transferase, an imp roved assay for CT was developed and used to follow its partial purifi cation. CT activity co-purifies with ACCase activity during gel permea tion chromatography. However, upon anion-exchange chromatography or na tive PAGE, CT separates from the BC and BCCP subunits of plastidial-AC Case and ACCase activity is lost. In addition, it is demonstrated that a previously sequenced pea chloroplast cDNA of unknown function (IEP9 6) with a predicted molecular weight of 91 kDa encodes the alpha-CT su bunit of the MS-ACCase. Antibodies raised against the first 404 amino acids of IEP96 protein detected a polypeptide with molecular weight of 91 kDa that co-eluted during gel permeation chromatography with plast idial CT and ACCase activities. These antibodies also immunoprecipitat ed the activities of both ACCase and CT with the concomitant precipita tion of the beta-CT subunit. Furthermore, antibodies against beta-CT i mmunoprecipitated the IEP96 protein. Two-dimensional PAGE and DEAE pur ification of ACCase protein demonstrated that the beta-CT forms a tigh t association with the IEP96 protein. Pea leaf was fractionated into s oluble and membrane fractions and the alpha-CT subunit was primarily a ssociated with the membrane fraction. Together, these data demonstrate that IEP96 is the alpha-CT subunit of pea chloroplast ACCase.