A major control point for the interaction among photosynthesis, photorespiration and mitochondrial respiration is the pyruvate dehydrogenase complex (PDC). The objectives of this research are to characterize events, mechanisms and modulators of PDC activity during development and photosynthesis in C3, C4, and CAM plants; extend biochemical characterization of PDC; establish in vitro and in situ regulatory mechanisms; examine the long term regulation of PDC; and study the expression of PDC's component enzymes, their import and complex assembly. We seek to establish conditions and metabolites that affect reversible phosphorylation of the mitochondrial PDC using purified, functional mitochondria coupled with inhibitors, altered metabolite levels and physiological environments. Antibodies, isotope labeling and enzyme activity measurements will be used to quantitate enzyme forms. Standard techniques will be used to clone to PDH components of PDC. Import and assembly studies will utilize our knowledge of a model protein and an Hsc70 chaperone system. This project will facilitate evaluation of the following concepts: 1) that reversible phosphorylation of PDC is a primary control mechanism for carbon entry into the Krebs cycle; 2) that PDC regulatory mechanisms differ during development and between photosynthetic and nonphotosynthetic tissues; 3) that the in vitro and in situ regulatory mechanisms function in vivo; 4) that import and assembly of PDC requires chaperones.
