Import, processing, and assembly of the alpha- and beta-subunits of chloroplast pyruvate dehydrogenase

Sequence comparisons were used to identify cDNAs potentially encoding the a lpha- and beta-subunits of chloroplast pyruvate dehydrogenase. Coupled in-v itro transcription plus translation was used to synthesize radiolabeled pyr uvate dehydrogenase alpha- and beta-subunit precursor proteins, When the pr ecursors were incubated with intact pea (Pisum sativum L.) seedling chlorop lasts in the presence of ATP, they were imported and proteolytically proces sed. In contrast, there was no import or processing of the precursors by is olated, intact pea seedling mitochondria. Monospecific antibodies to the re combinant pyruvate dehydrogenase alpha-subunit were additionally able to co -precipitate radiolabeled pyruvate dehydrogenase beta-subunit, indicating a ssociation between subunits after import and processing. Furthermore, size- exclusion chromatography was used to identify an alpha beta heterodimer tha t is an intermediate in the assembly of the native alpha 2 beta 2 heterotet rameric enzyme.