Submitted to: American Society of Plant Physiologists Meeting
Publication Type: Abstract Only
Publication Acceptance Date: 7/16/1999
Publication Date: N/A
Citation: Interpretive Summary:
Technical Abstract: An Arabidopsis thaliana cDNA encoding the dihydrolipoamide S-acetyltransferase (E2) subunit of the plastid pyruvate dehydrogenase complex was isolated from a lPRL2 cDNA 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 a single gene encodes plastid E2. The amino acid sequence has characteristic features of an acetyltransferase, namely, distinct lipoyl, subunit binding, and catalytic domains, though it is unusual in having only a single lipoyl domain. The in vitro synthesized plastid E2 precursor protein is Mr 67,000 on SDS-PAGE. Upon incubation of the precursor with pea chloroplasts, it was imported and processed to a mature size of Mr 60,000. The imported protein was located in the chloroplast stroma, associated with the endogenous pyruvate dehydrogenase (E1). Catalytically active recombinant plastid E2 was purified as a GST-fusion. Analysis of plastid E2 mRNA by RT-PCR 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 in leaves, and still less in siliques. Size exclusion chromatography was used to determine the size of the assembled recombinant E2. The plastid E2 is considerably different from the previously cloned mitochondrial isoform from A. thaliana.