CLONING AND CHARACTERIZATION OF THE DIHYDROLIPOAMIDE S-ACETYLTRANSFERASE (E2) SUBUNIT OF THE PLASTID PYRUVATE DEHYDROGENASE COMPLEX FROM ARABIDOPSIS THALIANA

Authors: MOONEY, BRIAN - UNIV OF MO, COLUMBIA, MO; Miernyk, Jan; RANDALL, DOUGLAS - UNIV OF MO, COLUMBIA, MO

Submitted to: Plant Physiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 10/21/1999
Publication Date: N/A
Citation: N/A

Interpretive Summary: In layman's terms, respiration is the use of energy to do work by living cells. Both growth and reproduction are affected by respiration. As a result, respiration must be carefully controlled or wasted energy would decrease crop yields and reduce agricultural productivity. The control of respiration in plant cells is a subject of ongoing study. A complex protein that is likely to be involved in an important control step in plant respiration was isolated and studied. Comparisons were made with a similar protein from animals and microbes, in order to identify which characteristics might be important in control of respiration. A method was developed to prepare large amounts of the plant protein that had full biological activity. This method will allow future studies of the gene or genes for this protein complex. Additionally, being able to produce large amounts of material will allow detailed analysis of the structures. This information will be important to researchers in their attempts to increase agricultural productivity by altering the control of plant cell respiration and to other plant scientists who will try to design more efficient crop plants through either classical breeding or biotechnology.

Technical Abstract: An Arabidopsis thaliana cDNA encoding the dihydrolipoamide S-acetyltransferase (E2) subunit of the plastid pyruvate dehydrogenase complex was isolated from a lambdaPRL2 library. The cDNA is 1709 bp in length, with a continuous open reading frame of 1440 bp encoding a protein of 480 amino acids and a molecular weight 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 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. This is the first report of a plastid E2.