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The pea chloroplast membrane‐associated protein, IEP96, is a subunit of acetyl‐CoA carboxylase
Author(s) -
Shorrosh Basil S.,
Savage Linda J.,
Soll Jürgen,
Ohlrogge John B.
Publication year - 1996
Publication title -
the plant journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.058
H-Index - 269
eISSN - 1365-313X
pISSN - 0960-7412
DOI - 10.1046/j.1365-313x.1996.10020261.x
Subject(s) - protein subunit , biochemistry , chloroplast , acetyl coa carboxylase , pisum , biology , pyruvate carboxylase , immunoprecipitation , enzyme , microbiology and biotechnology , gene
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 β‐carboxyltransferase (CT) subunits of the plastidial‐ACCase have recently been characterized and cloned. To further characterize the carboxyl‐transferase, 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 plastidiaI‐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 α‐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 β‐CT subunit. Furthermore, antibodies against β‐CT immunoprecipitated the IEP96 protein. Two‐dimensional PAGE and DEAE purification of ACCase protein demonstrated that the β‐CT forms a tight association with the IEP96 protein. Pea leaf was fractionated into soluble and membrane fractions and the α‐CT subunit was primarily associated with the membrane fraction. Together, these data demonstrate that IEP96 is the α‐CT subunit of pea chloroplast ACCase.