Cloning and Characterization of the Dihydrolipoamide S-Acetyltransferase Subunit of the Plastid Pyruvate Dehydrogenase Complex (E2) from Arabidopsis¹

Brian P. Mooney, Jan A. Miernyk, and Douglas D. Randall^*

Biochemistry Department, University of Missouri, Columbia, Missouri 65211 (B.P.M., J.A.M., D.D.R.); and Mycotoxin Research Unit, United States Department of Agriculture, Agricultural Research Service, National Center for Agricultural Utilization Research, Peoria, Illinois 61604 (J.A.M.)

An Arabidopsis cDNA encoding the dihydrolipoamide S-acetyltransferase subunit of the plastid pyruvate dehydrogenase complex (E2) was isolated from a PRL2 library. The cDNA is 1709 bp in length, with a continuous open reading frame of 1440 bp encoding a protein of 480 amino acids with a calculated molecular mass of 50,079 D. Southern analysis suggests that a single gene encodes plastid E2. The amino acid sequence has characteristic features of an acetyltransferase, namely, distinct lipoyl, subunit-binding, and catalytic domains, although it is unusual in having only a single lipoyl domain. The in vitro synthesized plastid E2 precursor protein has a relative molecular weight of 67,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Upon incubation of the precursor with pea (Pisum sativum) chloroplasts, it was imported and processed to a mature-sized relative molecular weight of 60,000. The imported protein was located in the chloroplast stroma, associated with the endogenous pyruvate dehydrogenase. Catalytically active recombinant plastid E2 was purified as a glutathione S-transferase fusion protein. Analysis of plastid E2 mRNA by reverse transcriptase-polymerase chain reaction showed highest expression in flowers, followed by leaves, siliques, and roots. The results of immunoblot analysis indicate that protein expression was similar in roots and flowers, less similar in leaves, and even less similar in siliques. This is the first report, to our knowledge, describing a plastid E2.

Plant Physiol. (1999) 120: 443-452
Copyright Clearance Center:   0032-0889/99/120//10
© 1999 American Society of Plant Physiologists

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