Cloning and characterization of the dihydrolipoamide S-acetyltransferase subunit of the plastid pyruvate dehydrogenase complex (E2) from arabidopsis

An Arabidopsis cDNA encoding the dihydrolipoamide S-acetyltransferase subun it of the plastid pyruvate dehydrogenase complex (E2) was isolated from a l ambda PRL2 library. The cDNA is 1709 bp in length, with a continuous open r eading frame of 1440 bp encoding a protein of 480 amino acids with a calcul ated molecular mass of 50,079 D. Southern analysis suggests that a single g ene encodes plastid E2. The amino acid sequence has characteristic features of an acetyltransferase, namely, distinct lipoyl, subunit-binding, and cat alytic domains, although it is unusual in having only a single lipoyl domai n. The in vitro synthesized plastid E2 precursor protein has a relative mol ecular weight of 67,000 on sodium dodecyl sulfate-polyacrylamide gel electr ophoresis. Upon incubation of the precursor with pea (Pisum sativum) chloro plasts, it was imported and processed to a mature-sized relative molecular weight of 60,000. The imported protein was located in the chloroplast strom a, associated with the endogenous pyruvate dehydrogenase. Catalytically act ive recombinant plastid E2 was purified as a glutathione S-transferase fusi on protein. Analysis of plastid E2 mRNA by reverse transcriptase-polymerase chain reaction showed highest expression in flowers, followed by leaves, s iliques, and roots. The results of immunoblot analysis indicate that protei n 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.