Import, processing, and assembly of the α- and β-subunits of chloroplast pyruvate dehydrogenase

Abstract.

 Sequence comparisons were used to identify cDNAs potentially encoding the α- and β-subunits of chloroplast pyruvate dehydrogenase. Coupled in-vitro transcription plus translation was used to synthesize radiolabeled pyruvate dehydrogenase α- and β-subunit precursor proteins. When the precursors were incubated with intact pea (Pisum sativum L.) seedling chloroplasts in the presence of ATP, they were imported and proteolytically processed. In contrast, there was no import or processing of the precursors by isolated, intact pea seedling mitochondria. Monospecific antibodies to the recombinant pyruvate dehydrogenase α-subunit were additionally able to co-precipitate radiolabeled pyruvate dehydrogenase β-subunit, indicating association between subunits after import and processing. Furthermore, size-exclusion chromatography was used to identify an αβ heterodimer that is an intermediate in the assembly of the native α2β2 heterotetrameric enzyme.