Precursor forms of the constituent polypeptides of pyruvate dehydrogenase and 2-oxoglutarate dehydrogenase complexes from ox heart

Abstract

The pyruvate dehydrogenase (PDC) and 2-oxoglutarate dehydrogenase (OGDC) multienzyme complexes of mitochondria are high-M, assemblies approximately the size of large and small ribosomal subunits, respectively. As all translation products of the mitochondrial genome have now been identified in higher animals (Chomyn et al . , 1985), i t is clear that the individual enzymes of the complex are nuclearcoded with a site of synthesis on cytoplasmic ribosomes (Suissa & Schatz, 1982). Thus, a complex sequence of molecular events must be involved in the biosynthesis, import, processing and assembly of these macromolecular aggregates including insertion of prosthetic groups, e.g. lipoic acid and FAD. Both these complexes are composed of multiple copies of three separate enzymes with a distinct lipoyl acyltransferase (E2) forming the structural core of each assembly. Initial decarboxylation of pyruvate or 2-oxoglutarate and reductive acylation of lipoyl groups on E2 is catalysed by a substrate-specific, thiamine diphosphate-requiring dehydrogenase, El. Production of the appropriate acyl-CoA derivative is promoted by E2 with subsequent re-oxidation of dihydrolipoamide groups by E3, lipoamide dehydrogenase (EC I .8.1.4), an FAD-linked homodimer, common to both complexes. Our approach to studying the initial events in the biosynthesis of PDC and OGDC has involved monitoring the appearance of cytoplasmic forms of these enzymes in vivo employing monospecific anitisera to the native complexes and each of the individual subunits. Immune replica analysis of a variety of cultured cells has established that three cell lines PK-15 (pig kidney), NBL-I (bovine kidney) and BRL (Buffalo rat liver) cells, exhibiting high rates of aerobic metabolism, contain substantial amounts of these complexes, approaching the levels in beef heart. In addition, immunoblotting has confirmed the monospecific nature of the antisera which can detect 5-50 ng of complex in crude SDS-extracts of whole cells. Detection of transient precursors is facilitated by inhibiting their uptake into the organelle with uncouplers of oxidative phosphorylation, e.g. 2,4-dinitrophenol (2 mM) or carbonyl cyanide trifluoromethoxyphenylhydrazone (1 0 PM) during a 4 h incubation with ['5S]methionine (Schleyer et al., 1982). Under these conditions, the mitochondrial electrochemical potential gradient (ApH+ ) is abolished completely with minimal effects (2&40%) on the overall rate of protein synthesis. Immunoprecipitations of ['5S]methionine-labelled cellular extracts were conducted in 1 O/ O (v/v) Triton X-100, 1 O/ O