323
conazole than wild-type cells, indicating that the native cellular membranes 4Wiseman 1994). It is tempting to
Erg11p activity was not fully restored. Overexpression speculate that the activated Erg3p-ER-HBD protein
of Pdr5p in these mutants exacerbated the growth de®- may not resemble the wild-type Erg3p, which might re-
ciency of the cells on ketoconazole. This strongly sug- sult in making these cells more resistant to tamoxifen.
gests that ketoconazole is exported from cells by Pdr5p,
In summary, our studies have shown that the activity
as has been reported earlier 4Sanglard et al. 1995). The of yeast proteins can post-translationally regulated by
enhanced export of ketoconazole results in a reduction the genomic fusion of the sequences encoding ER-HBD
in the intracellular concentration of the compound to a and the GR-HBD to the corresponding ORFs, using
level which is insucient to inhibit the amount of PCR-mediated, targeted integration. The expression of
Erg11p activity supplied by the Erg11p-HBD fusion the fusion proteins is driven by their own endogenous
protein. These mutants grew more slowly than wild-type promoters, enabling functional analysis under condi-
cells on ketoconazole. This observation again points to tions which resemble those in the wild-type situation.
the fact that dexamethasone and estradiol serve as sub- Importantly, the HBD-kanMx cassettes can be repeat-
strates for the Pdr5p pump 4Kralli et al. 1995; Mahe edly used for HBD tagging of several ORFs in a single
et al. 1996). Thus, the overexpression of Pdr5p leads to yeast strain by deletion of the kanMx marker via the
an enhanced eux of both steroids and ketoconazole, ¯anking two loxP sites 4Guldener et al. 1996). No cross-
resulting in the growth rates shown in Fig. 8.
reactivity between particular ER and GR ligands was
Using heterozygous diploid ERG11-HBD/ERG11 observed 4not shown), verifying that these ligands are
mutants we demonstrated that ERG11 has a haploin- highly speci®c for their cognate HBDs. We further
sucient phenotype: the loss of function of only one propose that the HBDs of the HSP-dependent receptors
ERG11 copy already leads to an abnormal phenotype for the steroid hormones progesterone, androgens and
4Giaever et al. 1999). This mutant grew signi®cantly mineralocorticoids should work in a similar way. With
more slowly than the homozygous wild-type cells on these tools it should be possible to dierentially regulate
medium containing ketoconazole, but faster than the the activities of up to ®ve dierent fusion proteins si-
haploid Erg11p-HBD-expressing strains 4not shown). multaneously in a single yeast strain simply by adding
Therefore, the HBD-mediated inactivation of protein the cognate ligands.
function also allows the modulation of protein activity
in terms of gene dosage.
The essential function of Erg11p was not completely
turned o on medium lacking ligands. This may be due
Acknowledgements We are grateful to Didier Picard for the gift of
plasmids p2HG/ER and pG/N795, Anand Bachhawat for plasmids
PDR5/YEplac195 and YEplac195, and Hans Hegemann for
plasmid pUG6. We thank Mark Johnston for critical reading and
to the localization of Erg11p in the membrane of the
endoplasmic reticulum. Nevertheless, we demonstrated
that this mutant is accessible to functional tests, e.g.
ketoconazole sensitivity. We therefore postulate that the
steroid-dependent activity of the Erg11p-HBD fusion
and certain other essential proteins allows one to obtain
viable cells which are more sensitive to particular com-
pounds or other assay conditions than wild-type cells.
The HBD-tagging method thus allows one to control
yeast protein function, even if these proteins are local-
ized in cellular compartments other than the cytoplasm
or the nucleus, as demonstrated with Erg11p and also
Erg3p.
comments to the manuscript. This work was supported by a grant
from the European Commission within the frame of EUROFAN II
4contract no. BIO4-CT97-2294).
References
Belli G, Gari E, Piedra®ta L, Aldea M, Herrero E 41998) An ac-
tivator/repressor dual system allows tight tetracycline-regulated
gene expression in budding yeast. Nucleic Acids Res 26: 942±
947
Burns N, Grimwade B, Ross-Macdonald PB, Choi EY, Finberg K,
Roeder GS, Snyder M 41994) Large-scale analysis of gene
expression, protein localization, and gene disruption in Sac-
charomyces cerevisiae. Genes Dev 8: 1087±1105
Daum G, Lees ND, Bard M, Dickson R 41998) Biochemistry, cell
biology and molecular biology of lipids of Saccharomyces
cerevisiae. Yeast 14: 1471±1510
The disruption of ERG3 results in a slow-growth
phenotype 4Palermo et al. 1997). Using the ER-HBG
tagging cassette we were able to mimic this phenotype on Dujon B 41996) The yeast genome project: what did we learn?
Trends Genet 12: 263±270
Entian K-D, et al 41999) Functional analyses of 150 deletion mu-
medium lacking steroids. The addition of tamoxifen led
to a complete restoration of normal growthkinetics, and
therefore to a functionally active Erg3p, whereas estra-
tants in Saccharomyces cerevisiae by a systematic approach.
Mol Gen Genet 262: 683±702
diol resulted only in partial rescue of Erg3p activity. This
could indicate that tamoxifen was more eective than
estradiol, which is unlikely, since tamoxifen rescued the
growthdefect of the mutant containing YDL139c-ER-
HBD only when added at higher concentrations. Alter-
natively, this eect may be speci®c for Erg3p, because
it has been shown, that erg3 mutants are resistant to
certain antifungal compounds, like ketoconazole and
nystatin 4Watson et al. 1988; Gachotte et al. 1996).
Tamoxifen also possesses antifungal activities, aecting
Gachotte D, Husselstein T, Bard M, Lacroute F, Benveniste P
41996) Isolation and characterization of an Arabidopsis thaliana
cDNA encoding a delta 7-sterol-C-5-desaturase by functional
complementation of a defective yeast mutant. Plant J 9: 391±
398
Giaever G, Shoemaker DD, Jones TW, Liang H, Winzeler EA,
Astromo A, Davis RW 41999) Genomic pro®ling of drug
sensitivities via induced haploinsuciency. Nat Genet 21: 278±
283
Gietz D, St Jean A, Woods RA, Schiestl RH 41992) Improved
method for high eciency transformation of intact yeast cells.
Nucleic Acids Res 20: 1425