Molecular and phenotypic characterization of yeast PDR1 mutants that show hyperactive transcription of various ABC multidrug transporter genes

Abstract

Mutations at the yeast PDR1 transcriptional regulator locus are responsible for overexpression of the three ABC transporter genes PDR5, SNQ2 and YOR1, associated with the appearance of multiple drug resistance. The nucleotide sequences of 13 alleles of PDR1, comprising 6 multidrug resistance mutants, 1 intragenic suppressor and 6 wild types, have been determined. Single amino acid substitutions were shown to result from the mutations pdr1-2 (M308I), pdr1-3 (F815S), pdr1-6 (K302Q), pdr1-7 (P298A) and pdr1-8 (L1036 W), whereas the intragenic suppressor mutant pdr1-100 is deleted for the two amino acids L537 and A538. An isogenic series of strains was constructed containing the mutant alleles pdr1-3, pdr1-6 and pdr1-8 integrated into the genome. We found that the levels of resistance to cycloheximide, oligomycin, 4-nitroquinoline-N-oxide and ketoconazole were increased in all three mutants. The increase was more pronounced in the pdr1-3 than in the pdr1-6 and pdr1-8 mutants. Studies of the activity of the promoters of the ABC genes PDR5, SNQ2 and YOR1 demonstrated that the combination of the PDR5 promoter and the pdr1-3 mutation resulted in the highest level of promoter induction. Concomitantly, the level of PDR5 mRNA, of Pdr5p protein, and of its associated nucleoside triphosphatase activity, was strongly increased in the plasma membranes of the PDR1 mutants. Again, the pdr1-3 allele was associated with a stronger effect than the pdr1-8 and pdr1-6 alleles. The locations of the mutations in the PDR1 gene indicate that at least three different regions distributed throughout the Pdr1p transcription factor may be mutated to generate a Pdr1p with considerably increased transcriptional activation potency. These gain-of-function mutations support the concept, recently proposed, that in members of the large family of yeast Zn2Cys6 transcription factors a central inhibitory domain exists (delineated by the pdr1-7, pdr1-6 and pdr1-2 mutations). This domain may interact in a locked conformation with a putative, more C-terminally located inhibitory domain (mutated in pdr1-3), and with the putative activation domain (mutated in pdr1-8).

Ssz1 Restores Endoplasmic Reticulum-Associated Protein Degradation in Cells Expressing Defective Cdc48–Ufd1–Npl4 Complex by Upregulating Cdc48

“…To determine whether pSSZ1 restored ERAD-M in cdc48-10 cells due to Ssz1p's role in PDR, we examined whether Pdr1p itself could restore ERAD. Indeed, when a plasmid expressing the Ssz1p-independent hyperactive PDR1-3 mutant (Carvajal et al 1997) was introduced into cdc48-10 cells, 6myc-Hmg2 degradation at 37°was markedly accelerated ( Figure 4A).…”

“…Ssz1p was overexpressed either as an untagged protein (pHE31; generously provided by E. Craig) or as an N-terminally HAtagged version (HA-Ssz1; generously provided by J. Frydman). The hyperactive Pdr1-3p mutant (F815S; Carvajal et al 1997), was expressed from plasmid pSK (generously provided by W. S. Moye-Rowley). Low-copy plasmids were used to express Rpn4p either as an untagged protein (p314CUP1RPN4) or as a C-terminally FLAG-tagged Rpn4 (p314CUP1RPN4-FLAG) (generously provided by Y. Xie).…”

Activation of the Multiple Drug Resistance Gene MDR1 in Fluconazole-Resistant, Clinical Candida albicans Strains Is Caused by Mutations in a trans -Regulatory Factor

“…However, as with the stable overexpression of the ABC transporters CDR1 and CDR2, the genetic basis for the constitutive activation of the MDR1 gene in such strains has not been elucidated. In Saccharomyces cerevisiae several regulatory proteins (Pdr1p, Pdr3p, and Yap1p) controlling expression of multiple drug resistance genes of the ABC transporter and major-facilitator superfamilies are known (1,3,5,11,12,15,16,20,28,30), and mutations in these regulators that result in upregulation of their respective target genes have been identified (4,19). Recently, functional homologues of these regulators have also been found in C. albicans (1,27); however, conflicting data about the roles of these transcriptional regulators have been obtained.…”

A Role for Ectophosphatase in Xenobiotic Resistance

“…Yeast cells must carefully regulate the ATP in their extracellular fluid to fully utilize either their endogenous MDR proteins (Kolaczkowski et al, 1996;Carvajal et al, 1997;Nourani et al, 1997) or those introduced by genetic manipulation.…”