EXPRESSION, PURIFICATION, AND CHARACTERIZATION OF THE DIHYDROLIPOAMIDE DEHYDROGENASE-BINDING PROTEIN OF THE PYRUVATE-DEHYDROGENASE COMPLEX FROM SACCHAROMYCES-CEREVISIAE

Genes encoding dihydrolipoamide dehydrogenase (E(3)) and the E(3)-binding protein (E(3)BP, protein X), components of the Saccharomyces cerevisiae pyruvate dehydrogenase (PDH) complex, were coexpressed in Escherichia coli to produce an E(3)BP-E(3) complex, thereby minimizing proteolysis of E(3)BP and facilitating its purification. The 2 genes were linked into a single transcriptional unit separated by a 31-nucleotide segment containing a ribosome-binding sequence. The E(3)BP-E(3) complex was highly purified and then separated into E(3) and E(3)BP by chromatography on hydroxylapatite in the presence of 5 M urea. The E(3)BP-E(3) complex combined rapidly with a pyruvate dehydrogenase (E(1))-dihydrolipoamide acetyltransferase (E(2)) subcomplex (E(1)-E(2) subcomplex) to reconstitute a functional PDH complex, with pyruvate oxidation activity similar to that of PDH complex from bakers' yeast. The stoichiometry of binding of E(3)BP and E3BP-E3 complex to the 60-subunit pentagonal dodecahedron-like E(2) was determined with a truncated form of E(2) (tE(2), residues 206-454) lacking the lipoyl domain and the E(1)-binding domain, and with E(1)-E(2) subcomplex, which contains intact E(2). Mixtures containing tE(2) or E(1)-E(2) subcomplex and excess E(3)BP or E(3)BP-E(3) complex were subjected to ultracentrifugation to separate the large complexes from unbound E(3)BP or E(3)BP-E(3), and the complexes were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis. After staining with Coomassie brilliant blue and destaining, the gels were analyzed with a video area densitometer. The results showed that the E(1)-E(2) subcomplex binds about 12 E(3)BP monomers attached to 12 E(3) homodimers. Similar results were obtained by analysis of highly purified PDH complex from bakers' yeast. Somewhat more E(3)BP (similar to 15 molecules) and E(3)BP-E(3) complex (similar to 14 molecules) bound to tE(2). Structural considerations suggest that 1 E(3)BP molecule, bearing and E(3) homodimer, is bound in each of the 12 faces of the pentagonal dodecahedron-like E(2).