CHARACTERIZATION OF PDH-BETA-1, THE STRUCTURAL GENE FOR THE PYRUVATE-DEHYDROGENASE BETA-SUBUNIT FROM SACCHAROMYCES-CEREVISIAE

The gene encoding the pyruvate dehydrogenase (PDH) beta subunit (E1beta) of the PDH complex from Saccharomyces cerevisiae has been cloned, sequenced, disrupted, and expressed. Two overlapping DNA fragments were generated from a yeast genomic DNA library by the polymerase chain reaction with synthetic oligonucleotide primers based on amino acid sequences of the yeast and human E1beta subunits. The DNA fragments were subcloned and sequenced. The composite sequence has an open reading frame of 1098 nucleotides encoding a putative presequence of 33 amino acid residues and a mature protein of 333 residues with a calculated M(r) = 36,486. Yeast and human E1beta exhibit 62% sequence identity. The size of the mRNA is almost-equal-to 1.5 kilobases. Hybridization analysis showed that the E1beta gene (PDHbeta1) is localized to chromosome II. Disruption of PDHbeta1 is not lethal under vegetative growth conditions. The null mutant transformed with PDHbeta1 on a unit-copy plasmid produced mature E1beta and a functional PDH complex.