Positive regulation of the LPD1 gene of Saccharomyces cerevisiae by the HAP2/HAP3/HAP4 activation system.
The LPD1 gene of Saccharomyces cerevisiae, encoding lipoamide dehydrogenase (LPDH), is subject to catabolite repression. The promoter of this gene contains a number of motifs for DNA-binding transcriptional activators, including three which show strong sequence homology to the core HAP2/HAP3/HAP4 binding motif. Here we report that transcription of LPD1 requires HAP2, HAP3 and HAP4 for release from glucose repression. In the wild-type strain, specific activity of LPDH was increased 12-fold by growth on lactate, 10-fold on glycerol and four- to five-fold on galactose or raffinose, compared to growth on glucose. In hap2, hap3 and hap4 null mutants, the specific activities of LPDH in cultures grown on galactose and raffinose showed only slight induction above the basal level on glucose medium. Similar results were obtained upon assaying for beta-galactosidase production in wild-type, or hap2, hap3 or hap4 mutant strains carrying a single copy of the LPD1 promoter fused in frame to the lacZ gene of Escherichia coli and integrated at the URA3 locus. Transcript analysis in wild-type and hap2 mutants confirmed that the HAP2 protein regulates LPD1 expression at the level of transcription in the same way as it does for the CYC1 gene. Site-directed mutagenesis of the putative HAP2/HAP3/HAP4 binding site at -204 relative to the ATG start codon showed that this element was required for full derepression of the LPD1 gene on non-fermentable substrates.
Bowman
S B
SB
School of Biochemistry, University of New South Wales, Kensington, Australia.
Zaman
Z
Z
Collinson
L P
LP
Brown
A J
AJ
Dawes
I W
IW
D001483:Base Sequence; D001665:Binding Sites; D016384:Consensus Sequence; D003574:Cytochrome c Group / Q000096:biosynthesis / Q000235:genetics; D004268:DNA-Binding Proteins / Q000096:biosynthesis / Q000235:genetics; D008058:Dihydrolipoamide Dehydrogenase / Q000096:biosynthesis / Q000235:genetics*; D004789:Enzyme Activation; D004794:Enzyme Repression; D005656:Fungal Proteins / Q000096:biosynthesis / Q000235:genetics*; D015966:Gene Expression Regulation, Fungal; D005800:Genes, Fungal; D008969:Molecular Sequence Data; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000254:growth & development; D015534:Trans-Activators / Q000235:genetics; D014158:Transcription, Genetic
D003574:Cytochrome c Group; D004268:DNA-Binding Proteins; D005656:Fungal Proteins; D015534:Trans-Activators; D008058:Dihydrolipoamide Dehydrogenase