Transcriptional activation by yeast PDR1p is inhibited by its association with NGG1p/ADA3p.
NGG1p/ADA3p forms a coactivator/repressor complex (ADA complex) in association with at least two other yeast proteins, ADA2p and GCN5p, that is involved in regulating transcriptional activator proteins including GAL4p and GCN4p. Using a two-hybrid analysis, we found that the carboxyl-terminal transcriptional activation domain of PDR1p, the primary regulatory protein involved in yeast pleiotropic drug resistance, interacts with the amino-terminal 373 amino acids of NGG1p (NGG1p1-373). This interaction was confirmed by coimmunoprecipitation of epitope-tagged derivatives of NGG1p and PDR1p from crude extracts. An overlapping region of the related transcriptional activator PDR3p was also found to interact with NGG1p. Amino acids 274-307 of NGG1p were required for interaction with PDR1p. This same region is required for inhibition of transcriptional activation by GAL4p. The association between NGG1p1-373 and PDR1p may be indirect, possibly mediated by the ADA complex since the two-hybrid interaction required the presence of full-length NGG1. A partial requirement for ADA2 was also found. This suggests that an additional component of the ADA complex, regulated by ADA2p, may mediate the interaction. Transcriptional activation by a GAL4p DNA binding domain fusion of PDR1p was enhanced in ngg1 and ada2 disruption strains. Similar to its action on GAL4p, the ADA complex acts to inhibit the activation domain of PDR1p.
Martens
J A
JA
Department of Biochemistry, University of Western Ontario, London, Ontario, Canada N6A 5C1.
Genereaux
J
J
Saleh
A
A
Brandl
C J
CJ
D000595:Amino Acid Sequence; D004268:DNA-Binding Proteins / Q000737:chemistry; D004352:Drug Resistance, Microbial; D005656:Fungal Proteins / Q000378:metabolism; D008969:Molecular Sequence Data; D016296:Mutagenesis; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012097:Repressor Proteins / Q000302:isolation & purification / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins; D017384:Sequence Deletion; D017386:Sequence Homology, Amino Acid; D015534:Trans-Activators; D014157:Transcription Factors / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D015533:Transcriptional Activation; D001616:beta-Galactosidase / Q000378:metabolism
D004268:DNA-Binding Proteins; D005656:Fungal Proteins; C083323:NGG1 protein, S cerevisiae; C085480:PDR1 protein, S cerevisiae; C089261:PDR3 protein, S cerevisiae; D011993:Recombinant Fusion Proteins; D012097:Repressor Proteins; D029701:Saccharomyces cerevisiae Proteins; D015534:Trans-Activators; D014157:Transcription Factors; D001616:beta-Galactosidase