A major point of interaction among photosynthesis, photorespiration and mitochondrial respiration is the reaction catalyzed by the mitochondrial pyruvate dehydrogenase complex (mtPDC). This multi-enzyme complex is the primary entry point for carbon into the Krebs cycle for energy production and the generation of biosynthetic intermediates. The plastid isoform of PDC provides the precursor for fatty acid biosynthesis, branched-chain amino acids, some isoprenoids and growth regulators. The mtPDC is regulated in part by reversible phosphorylation of multiple serine residues catalyzed by a novel and specific histidine-like protein kinase (PDK). Objectives: To establish the phosphorylation sites on the E1 subunits of plant PDC; to characterize recombinant PDKs with regard to effectors and kinetics; to establish the expression of PDK isoforms as function of plant development and environmental influences; to establish amino acids essential for catalysis and binding in the PDK primary sequence; to establish the physiological role of PDKs using transgenic plants over- and underexpressing PDK and by identifying "gene knockouts" of PDK in maize and Arabidopsis thaliana; to characterize events, mechanisms and modulators of PDC activity during development and photosynthesis in C4 and C3 plants; to investigate in vitro assembly of PDC component enzymes and their import and assembly. Experimental approach: 1) 32P-labeled E1 subunit will be purified from the complexes exhibiting various states of activity and the phosphorylated serines identified. 2) Effectors and kinetics of recombinant PDK will be determined. 3) Specific hybridization probes and antibodies for PDK isoforms will be used to measure developmental and spatial expression, and responses to environmental and nutritional cues. 4) Standard site-directed mutagenesis methods will be used to systematically change specific amino acid residues of PDK, and activities will be assayed using kinase-depleted PDC. 5) A. thaliana will be transformed; down regulation of PDK and PDH will be accomplished using T-DNA tagged genomic DNA pools. 6) In vitro assembly studies will utilize subunits of component enzymes in conjunction with various chaperone systems; import and assembly will utilize purified pea mitochondria and subunit precursors. 7) Conditions and metabolites that affect reversible phosphorylation of mtPDC will be determined using purified functional mitochondria, protoplasts or whole tissues. Significance: The results obtained will help clarify control of interactions among multiple distinct catabolic and anabolic pathways. The mechanisms and structural requirements of a unique class of protein kinases will be determined. Factors that affect expression of PDK and of the PDC subunits will be identified. Details of the synthesis, organellar import, and assembly of the components of PDC will be resolved. Finally, mechanisms for controlling the activities of plastid and mtPDC will be ascertained. Plastid PDC is likely to be essential for fatty acid biosynthesis, and unique regulatory properties are required in this subcellular compartment.
