We have compared Saccharomyces cerevisiae global gene expression in wild-type and mutants (Δhap2 and Δhap4) of the HAP transcriptional complex, which has been shown to be necessary for growth on respiratory substrates. Several hundred ORFs are under positive or negative control of this complex and we analyse here in detail the effect of HAP on mitochondria. We found that most of the genes upregulated in the wild-type strain were involved in organelle functions, but practically none of the downregulated ones. Nuclear genes encoding the different subunits of the respiratory chain complexes figure in the genes more expressed in the wild-type than in the mutants, as expected, but in this group we also found key components of the mitochondrial translation apparatus. This control of mitochondrial translation may be one of the means of coordinating mitochondrial and nuclear gene expression in elaborating the respiratory chain. In addition, HAP controls the nuclear genes involved in several other mitochondrial processes (import, mitochondrial division) that define the metabolic state of the cell, but not mitochondrial DNA replication and transcription. In most cases, a putative CCAAT-binding site is present upstream of the ORF, while in others no such sites are present, suggesting the control to be indirect. The large number of genes regulated by the HAP complex, as well as the fact that HAP also regulates some putative transcriptional activators of unknown function, place this complex at a hierarchically high position in the global transcriptional regulation of the cell.

The effects of fenpropimorph on sterol biosynthesis and growth of Saccharomyces cerevisiae were examined to pinpoint the mode of action of fungicides that inhibit ergosterol biosynthesis. Taking advantage of sterol auxotrophy and sterol permeability in mutant strains, we show that growth inhibition is strongly correlated with inhibition of sterol biosynthesis. We confirm that in vivo and at low concentrations, fenpropimorph inhibits A8-*A7-sterol isomerase, and in addition, when it is used at higher concentrations, it inhibits A14-sterol reductase. We show also that the fungistatic effect of fenpropimorph is not due to the accumulation of abnormal sterols in treated cells but is linked to the specific inhibition of ergosterol biosynthesis, leading to the arrest of cell proliferation in the unbudded G1 phase of the cell cycle.

We have transformed Saccharomyces cerevisiae with a genomic library contained in the replicative vector pFL44. The resulting transformants were screened for resistance to fenpropidin, a specific inhibitor of sterol 14-reductase. A plasmid was isolated that transformed yeast both to resistance to fenpropidin and to an increased specific activity of sterol 14-reductase. Sterol analysis of transformed cells grown in the presence of increasing concentrations of the inhibitor confirmed that resistance was a consequence of over-production of sterol 14-reductase. By chromosomal gene disruption, we have, for the first time, constructed yeast strains defective in sterol 14-reductase. As expected, since yeast in unable to take up sterols in aerobiosis, the disrupted strains do not grow in the presence of oxygen, even if exogenous sterols are supplied. However, disrupted cells grow in anaerobiosis with exogenous oleic acid and ergosterol supplements. They also grow in aerobiosis if they bear an additional mutation allowing sterol uptake. In this last growth condition the cells require a "sparking" ergosterol supplementation (25 nM) and accumulate ignosterol (ergosta-8,14-dienol) as the end-product of the sterol pathway. These results reveal that ignosterol is not obviously toxic to yeast membranes and strongly suggest that the molecular basis of the antifungal-activity morpholine and piperidine is directly related to the specific inhibition of ergosterol formation.

Exogenous sterols do not permeate wild-type Saccharomyces cerevisiae in aerobic conditions. However, mutant strain FKerg7, affected in lanosterol synthase, is a sterol auxotroph which is able to grow aerobically in the presence of ergosterol. Viability of this strain depends on the presence of an additional mutation, aux30, that leads to sterol permeability. Cells bearing the aux30 mutation fail to grow in standard yeast nitrogen base medium containing pyridoxine but grow normally if pyridoxine is replaced by either pyridoxal or pyridoxamine.

Screening for resistance to fenpropimorph was undertaken in order to isolate yeast mutants affected in the regulation of the ergosterol pathway. Among the mutants isolated, one bearing the recessive fen1-1 mutation was characterized by a 1.5-fold increase in the ergosterol level and a general resistance to sterol biosynthesis inhibitors. The fen1-1 mutation was linked to MAT locus on chromosome III. The measurement of enzyme activities involved in the ergosterol pathway revealed that isopentenyl diphosphate (IPP) isomerase activity was specifically increased 1.5-fold as compared to the wild type strain. However, overexpression of IPP isomerase in the wild type strain was not by itself sufficient to lead to sterol increase or resistance to sterol biosynthesis inhibitors, showing that IPP isomerase is not a limiting step in the pathway. The fen1-1 mutation permits viability in aerobiosis of yeast disrupted for sterol-14 reductase in absence of exogenous ergosterol supplementation, whereas the corresponding strain bearing the wild type FEN1 allele grows only in anaerobiosis. This result shows that ignosterol is able to efficiently replace ergosterol as bulk membrane component and that the fen1-1 mutation eliminates the specific ergosterol requirement in yeast.