Recently we deleted the pyruvate decarboxylase structural gene PDCl from the genome of the yeast Saccharomyces cerevisiae. The pdcl deletion mutants had pyruvate decarboxylase activity due to the presence of a second structural gene [Schaaff, I., Green, J. B. A,, Gozalbo, D. & Hohmann, S. (1989) Curr. Genet. 15, [75][76][77][78][79][80][81]. We cloned and sequenced this gene which we call PDCS. The predicted amino acid sequences of PDCl and PDCS are 88% identical.Deletion of PDCS did not cause any decrease in the specific pyruvate decarboxylase activity whilepdcl deletion mutants had 80% of the wild-type activity. Deletion mutants lacking both PDCl and PDCS did not show any detectable pyruvate decarboxylase activity in vitro and were unable to ferment glucose. This indicates that PDCl and PDCS are the only structural genes for pyruvate decarboxylase in yeast.The PDCS isoenzyme showed a slightly higher K , value for its substrate pyruvate than the PDCI product (PDCS: K, = 8 mM; PDCI : K, = 5 mM), as measured in crude extract of pdcl and pdc5 deletion mutants, respectively.PDCS is only expressed in pdcl deletion mutants. No mRNA transcribed from PDCS could be detected in wild-type cells. Thus, in addition to the control by glucose induction, pyruvate decarboxylase activity seems to be subject to autoregulation. Similar phenomena have been described previously for tubulin, histones and a ribosomal protein but not for metabolic enzymes.Pyruvate decarboxylase is the key enzyme of alcoholic fermentation since it cleaves pyruvate, the end product of glycolysis, to CO, and acetaldehyde [1].
.2).Note. The nucleotide sequence of the PDCS DNA sequence published here has been deposited with the EMBL/GenBank/DDBJ Nucleotide Sequence data bank and will be availablc under the accession number XI 5668. Surprisingly, a pdcl deletion mutation conferred a different phenotype than thepdcl-8 mutation described by Schmitt and Zimmermann [7]. The deletion mutants fermented glucose but they had about 50% of the wild-type pyruvate decarboxylase activity [12]. This activity was most probably due to a second gene encoding pyruvate decarboxylase which is very similar to PDCI since the pdcl deletion mutants still had a full-length mRNA hybridizing to PDCl probes in Northern blot analysis [12].Here we report the cloning and molecular analysis of PDC5, the second pyruvate decarboxylase structural gene. This gene seems to be not, or only poorly, expressed in wildtype cells but is activated in the pdcl deletion mutants.
MATERIALS AND METHODS
Yeast strainsStrain 1.1.-6B