Loss-of-functionpdr3mutations convert the Pdr3p transcription activator to a protein suppressing multidrug resistance inSaccharomyces cerevisiae

Abstract

The PDR1 and PDR3 genes encode the main transcription activators involved in the control of multidrug resistance in Saccharomyces cerevisiae. To identify the amino acids essential for Pdr3p function, the loss-of-function pdr3 mutants were isolated and characterized. Two plasmid-borne pdr3 alleles, pdr3-E902Ter and pdr3-D853Y, which failed to complement drug hypersensitivity in the Deltapdr1Deltapdr3 mutant strain, were isolated. The E902Ter mutation resulted in a truncated protein lacking the C-terminal activation domain. The D853Y mutation allowed the expression of entire Pdr3p, but its transactivation function was lost. When overexpressed from the P(GAL1) promoter, the two mutant alleles increased the sensitivity of wild-type cells to cycloheximide and fluconazole and suppressed drug resistance in gain-of-function pdr1 and pdr3 mutant strains. The drug-sensitizing effect of overexpressed loss-of-function pdr3 mutant alleles correlated with their ability to suppress PDR5 transcription and rhodamine 6G accumulation in transformants of the wild-type and Deltapdr1 mutant strains. These results demonstrate that amino acid residue Asp853 is essential for Pdr3p function, and indicate that specific loss-of-function pdr3 mutations can convert the Pdr3p transcription activator to a multicopy suppressor of multidrug resistance.

Site‐directed mutagenesis of Asp853 in Pdr3p transcriptional activator from Saccharomyces cerevisiae

“…detrimental effect of Tyr853 on biological activity of Pdr3p previously observed in the pdr3-D853Y single mutant (Sidorova et al, 2007).…”

“…PDR5 expression, drug resistance and rhodamine 6G efflux in pdr3 double mutants Recently, the multicopy plasmid-borne loss-offunction pdr3-D853Y mutant allele has been found to act as a suppressor of multidrug resistance in gain-of-function chromosomal pdr1 and pdr3 mutants (Sidorova et al, 2007). To reveal whether the D853Y mutation occurring in the same protein with gain-of-function pdr3 mutation affects the transactivation function of Pdr3p, the two double mutants were constructed using site-directed mutagenesis.…”

“…Phenotypes of yeast transformants harbouring plasmid-borne pdr3 mutations were determined and compared with those containing the wildtype PDR3 gene and loss-of-function pdr3-D853Y allele. Since the D853Y amino acid substitution suppressed the transactivation function of Pdr3p (Sidorova et al, 2007), this mutation in the cisposition was also introduced into the two gainof-function pdr3-7 and pdr3-18 alleles, containing G253E (Nourani et al, 1997) and G834S (Simonics et al, 2000) mutations, respectively. …”

“…Plasmids pFL38-PDR3, pFL38-pdr3-7 (Nourani et al, 1997), pFL38-pdr3-18 (Simonics et al, 2000), pFL38-pdr3-Site-directed mutagenesis of Asp853 in Pdr3p 279 D853Y (Sidorova et al, 2007) and pYES2-PDR3 (Kozovska et al, 2004) served as sources of the PDR3 alleles. Mutations were introduced into pYES2-PDR3 from pFL38-borne pdr3 mutant alleles by exchanging the 2308 bp PacI-XhoI DNA fragment in the PDR3 gene.…”

“…One allele, pdr3-E902Ter, encoded the truncated Pdr3p, devoid of C-terminal activation domain, and another one was coding for Pdr3p containing the Asp853Tyr amino acid substitution (Sidorova et al, 2007). Along with a loss of transactivation activity, both mutant proteins gained a new function; they acted as multicopy suppressors of multidrug resistance in yeast (Sidorova et al, 2007).…”