The pea chloroplast membrane-associated protein, IEP96, is a subunit of acetyl-CoA carboxylase.
Two forms of acetyl-CoA carboxylase (ACCase) have been characterized in 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 characterized and cloned. To further characterize the carboxyltransferase, an improved assay for CT was developed and used to follow its partial purification. CT activity co-purifies with ACCase activity during gel permeation chromatography. However, upon anion-exchange chromatography or native PAGE, CT separates from the BC and BCCP subunits of plastidial-ACCase and ACCase activity is lost. In addition, it is demonstrated that a previously sequenced pea chloroplast cDNA of unknown function (IEP96) with a predicted molecular weight of 91 kDa encodes the alpha-CT subunit 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 plastidial CT and ACCase activities. These antibodies also immunoprecipitated the activities of both ACCase and CT with the concomitant precipitation of the beta-CT subunit. Furthermore, antibodies against beta-CT immunoprecipitated the IEP96 protein. Two-dimensional PAGE and DEAE purification of ACCase protein demonstrated that the beta-CT forms a tight association with the IEP96 protein. Pea leaf was fractionated into soluble and membrane fractions and the alpha-CT subunit was primarily associated with the membrane fraction. Together, these data demonstrate that IEP96 is the alpha-CT subunit of pea chloroplast ACCase.
Shorrosh
B S
BS
Department of Botany and Plant Pathology, Michigan State University, East Lansing 48824-1321, USA.
Savage
L J
LJ
Soll
J
J
Ohlrogge
J B
JB
D000103:Acetyl-CoA Carboxylase / Q000737:chemistry* / Q000276:immunology; D000818:Animals; D000906:Antibodies; D060365:Chloroplast Proteins; D002736:Chloroplasts / Q000737:chemistry* / Q000201:enzymology*; D008565:Membrane Proteins / Q000737:chemistry* / Q000276:immunology; D015394:Molecular Structure; D008970:Molecular Weight; D018532:Pisum sativum / Q000737:chemistry / Q000201:enzymology; D010940:Plant Proteins / Q000737:chemistry* / Q000276:immunology; D011487:Protein Conformation; D011817:Rabbits
D000906:Antibodies; D060365:Chloroplast Proteins; D008565:Membrane Proteins; D010940:Plant Proteins; D000103:Acetyl-CoA Carboxylase