Bax is a pro‐apoptotic member of the Bcl‐2 family of proteins involved in the regulation of genetically programmed cell death in mammalian cells. It has been shown that heterologous expression of Bax in several yeast species, such as Saccharomyces cerevisiae, Schizosaccharomyces pombe and Pichia pastoris, also induces cell death. In this study we investigated the effects of Bax expression in the pathogenic yeast Candida albicans . Cell death inducing expression of Bax required a synthetic BAX gene that was codon‐optimized for expression in Candida albicans . Expression of this BAX gene resulted in growth inhibition and cell death. By fusing Bax with the yeast enhanced green fluorescent protein of Aequoria victoria , the cell death‐inducing effect of Bax was increased due to reduced proteolytic degradation of Bax. Using this fusion protein we showed that, upon expression in C. albicans , Bax co‐localizes with the mitochondria. Furthermore, we showed for the first time that expression of Bax in yeast causes the mitochondria, which are normally distributed throughout the cell, to cluster in the perinuclear region. Copyright © 2004 John Wiley & Sons, Ltd.

In the yeast, Saccharomyces cerevisiae, pyruvate decarboxylase (Pdc) is encoded by the two isogenes PDC1 and PDC5. Deletion of the more strongly expressed PDC1 gene stimulates the promoter activity of both PDC1 and PDC5, a phenomenon called Pdc autoregulation. Hence, pdc1Delta strains have high Pdc specific activity and can grow on glucose medium. In this work we have characterized the mutant alleles pdc1-8 and pdc1-14, which cause strongly diminished Pdc activity and an inability to grow on glucose. Both mutant alleles are expressed as detectable proteins, each of which differs from the wild-type by a single amino acid. The cloned pdc1-8 and pdc1-14 alleles, as well as the in-vitro-generated pdc1-51 (Glu51Ala) allele, repressed expression of PDC5 and diminished Pdc specific activity. Thus, the repressive effect of Pdc1p on PDC5 expression seems to be independent of its catalytic activity. A pdc1-8 mutant was used to isolate spontaneous suppressor mutations, which allowed expression of PDC5. All three mutants characterized had additional mutations within the pdc1-8 allele. Two of these mutations resulted in a premature translational stop conferring phenotypes virtually indistinguishable from those of a pdc1Delta mutation. The third mutation, pdc1-803, led to a deletion of two amino acids adjacent to the pdc1-8 mutation. The alleles pdc1-8 and pdc1-803 were expressed in Escherichia coli and purified to homogeneity. In the crude extract, both proteins had 10% residual activity, which was lost during purification, probably due to dissociation of the cofactor thiamin diphosphate (ThDP). The defect in pdc1-8 (Asp291Asn) and the two amino acids deleted in pdc1-803 (Ser296 and Phe297) are located within a flexible loop in the beta domain. This domain appears to determine the relative orientation of the alpha and gamma domains, which bind ThDP. Alterations in this loop may also affect the conformational change upon substrate binding. The mutation in pdc1-14 (Ser455Phe) is located within the ThDP fold and is likely to affect binding and/or orientation of the cofactor in the protein. We suggest that autoregulation is triggered by a certain conformation of Pdc1p and that the mutations in pdc1-8 and pdc1-14 may lock Pdc1p in vivo in a conformational state which leads to repression of PDC5.

We investigated the importance of the interaction between the Nl'-atom of the cofactor thiamine diphosphate and glutamic acid residue 51 in pyruvate decarboxylase (EC 4.1. 1.1). The yeast wild type gene PDCl and the respective mutant genes (E51Q and E51A) were expressed in Escherichia coli. The three enzymes were purified to homogeneity. They comigrated as a single band during silver-stained SDS/PAGE with a molecular mass of 60 000 Da. A molecular mass of 61 200 +/- 200 Da was determined by mass spectrometry for the subunit. The native enzyme is a homotetramer as demonstrated by gel filtration experiments. Near- and far-UV CD spectra showed no significant differences for the apoenzyme of the wild type and the mutants. Slight differences in the rate of thiamine diphosphate binding to the apoprotein component were observed between the wild type and the E51Q PDC by CD spectroscopy. Compared to the wild type enzyme, thiamine diphosphate binding at the E51A mutant apoprotein is very slow. Only 0.04% of the catalytic activity of the wild type enzyme was observed for the E51Q mutant; the E51A mutant has no detectable catalytic activity. The S0.5 value for the substrate pyruvate is increased 33-fold for the E51Q mutant. Substrate activation was observed for both the wild type and the E51Q mutant. The interaction between the N1'-atom of the coenzyme and glutamic acid 51 strongly influences the catalytic activity but only moderately the binding of the cofactor to the apoenzyme and the substrate activation rate.