scholarly journals The Karyopherin Kap122p/Pdr6p Imports Both Subunits of the Transcription Factor Iia into the Nucleus

1999 ◽  
Vol 147 (2) ◽  
pp. 235-246 ◽  
Author(s):  
Anton A. Titov ◽  
Günter Blobel
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Drug Resistance ◽  
Transcription Factor ◽  
Recombinant Proteins ◽  
Nuclear Import ◽  
Small Subunit ◽  
Cytoplasmic Localization ◽  
Pleiotropic Drug Resistance ◽  
Wild Type ◽  
General Transcription Factor

We discovered a nuclear import pathway mediated by the product of the previously identified Saccharomyces cerevisiae gene PDR6 (pleiotropic drug resistance). This gene product functions as a karyopherin (Kap) for nuclear import. Consistent with previously proposed nomenclature, we have renamed this gene KAP122. Kap122p was localized both to the cytoplasm and the nucleus. As a prominent import substrate of Kap122p, we identified the complex of the large and small subunit (Toa1p and Toa2p, respectively) of the general transcription factor IIA (TFIIA). Recombinant GST-Kap122p formed a complex with recombinant His6-Toa1p/Toa2p. In wild-type cells, Toa1p and Toa2p were localized to the nucleus. Consistent with Kap122p being the principal Kap for import of the Toa1p–Toa2p complex, we found that deletion of KAP122 results in increased cytoplasmic localization of both Toa1p and Toa2p. Deletion of KAP122 is not lethal, although deletion of TOA1 and TOA2 is. Together these data suggest that Kap122p is the major Kap for the import of Toa1p–Toa2p into the nucleus. Like other substrate–Kap complexes, the Toa1p/Toa2p/Kap122p complex isolated from yeast cytosol or reconstituted from recombinant proteins, was dissociated by RanGTP but not RanGDP. Kap122p bound to nucleoporins, specifically, to the peptide repeat–containing fragments of Nup1p and Nup2p.

scholarly journals RPD3 and UME6 are involved in the activation of PDR5 transcription and pleiotropic drug resistance in ρ0 cells of Saccharomyces cerevisiae

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Yoichi Yamada
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Drug Resistance ◽  
Positive Response ◽  
Mrna Levels ◽  
Transporter Gene ◽  
Transcriptional Expression ◽  
Pleiotropic Drug Resistance ◽  
Wild Type ◽  
Binding Partners ◽  
Retrograde Signalling

Abstract Background In Saccharomyces cerevisiae, the retrograde signalling pathway is activated in ρ0/− cells, which lack mitochondrial DNA. Within this pathway, the activation of the transcription factor Pdr3 induces transcription of the ATP-binding cassette (ABC) transporter gene, PDR5, and causes pleiotropic drug resistance (PDR). Although a histone deacetylase, Rpd3, is also required for cycloheximide resistance in ρ0/− cells, it is currently unknown whether Rpd3 and its DNA binding partners, Ume6 and Ash1, are involved in the activation of PDR5 transcription and PDR in ρ0/− cells. This study investigated the roles of RPD3, UME6, and ASH1 in the activation of PDR5 transcription and PDR by retrograde signalling in ρ0 cells. Results ρ0 cells in the rpd3∆ and ume6∆ strains, with the exception of the ash1∆ strain, were sensitive to fluconazole and cycloheximide. The PDR5 mRNA levels in ρ0 cells of the rpd3∆ and ume6∆ strains were significantly reduced compared to the wild-type and ash1∆ strain. Transcriptional expression of PDR5 was reduced in cycloheximide-exposed and unexposed ρ0 cells of the ume6∆ strain; the transcriptional positive response of PDR5 to cycloheximide exposure was also impaired in this strain. Conclusions RPD3 and UME6 are responsible for enhanced PDR5 mRNA levels and PDR by retrograde signalling in ρ0 cells of S. cerevisiae.

scholarly journals Involvement of the Pleiotropic Drug Resistance Response, Protein Kinase C Signaling, and Altered Zinc Homeostasis in Resistance of Saccharomyces cerevisiae to Diclofenac

2011 ◽  
Vol 77 (17) ◽  
pp. 5973-5980 ◽  
Author(s):  
Jolanda S. van Leeuwen ◽  
Nico P. E. Vermeulen ◽  
J. Chris Vos
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Drug Resistance ◽  
Cell Wall ◽  
Transcription Factor ◽  
Protein Kinase C ◽  
Protein Kinase ◽  
Zinc Homeostasis ◽  
Yeast Cells ◽  
Pleiotropic Drug Resistance ◽  
Kinase C

ABSTRACTDiclofenac is a widely used analgesic drug that can cause serious adverse drug reactions. We usedSaccharomyces cerevisiaeas a model eukaryote with which to elucidate the molecular mechanisms of diclofenac toxicity and resistance. Although most yeast cells died during the initial diclofenac treatment, some survived and started growing again. Microarray analysis of the adapted cells identified three major processes involved in diclofenac detoxification and tolerance. In particular, pleiotropic drug resistance (PDR) genes and genes under the control of Rlm1p, a transcription factor in the protein kinase C (PKC) pathway, were upregulated in diclofenac-adapted cells. We tested if these processes or pathways were directly involved in diclofenac toxicity or resistance. Of the pleiotropic drug resistance gene products, the multidrug transporter Pdr5p was crucially important for diclofenac tolerance. Furthermore, deletion of components of the cell wall stress-responsive PKC pathway increased diclofenac toxicity, whereas incubation of cells with the cell wall stressor calcofluor white before the addition of diclofenac decreased its toxicity. Also, diclofenac induced flocculation, which might trigger the cell wall alterations. Genes involved in ribosome biogenesis and rRNA processing were downregulated, as were zinc-responsive genes. Paradoxically, deletion of the zinc-responsive transcription factor Zap1p or addition of the zinc chelator 1,10-phenanthroline significantly increased diclofenac toxicity, establishing a regulatory role for zinc in diclofenac resistance. In conclusion, we have identified three new pathways involved in diclofenac tolerance in yeast, namely, Pdr5p as the main contributor to the PDR response, cell wall signaling via the PKC pathway, and zinc homeostasis, regulated by Zap1p.

scholarly journals Regulation of Transcription Factor Pdr1p Function by an Hsp70 Protein in Saccharomyces cerevisiae

1998 ◽  
Vol 18 (3) ◽  
pp. 1147-1155 ◽  
Author(s):  
Timothy C. Hallstrom ◽  
David J. Katzmann ◽  
Rodrigo J. Torres ◽  
W. John Sharp ◽  
W. Scott Moye-Rowley
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Drug Resistance ◽  
Transcription Factor ◽  
Regulatory Protein ◽  
Atp Binding ◽  
Regulation Of Transcription ◽  
Pleiotropic Drug Resistance ◽  
Atp Binding Cassette Transporter ◽  
Encoding Genes ◽  
Atp Binding Cassette

ABSTRACT Multiple or pleiotropic drug resistance in the yeastSaccharomyces cerevisiae requires the expression of several ATP binding cassette transporter-encoding genes under the control of the zinc finger-containing transcription factor Pdr1p. The ATP binding cassette transporter-encoding genes regulated by Pdr1p include PDR5 and YOR1, which are required for normal cycloheximide and oligomycin tolerances, respectively. We have isolated a new member of the PDR gene family that encodes a member of the Hsp70 family of proteins found in this organism. This gene has been designated PDR13 and is required for normal growth. Overexpression of Pdr13p leads to an increase in both the expression of PDR5 and YOR1 and a corresponding enhancement in drug resistance. Pdr13p requires the presence of both the PDR1 structural gene and the Pdr1p binding sites in target promoters to mediate its effect on drug resistance and gene expression. A dominant, gain-of-function mutant allele ofPDR13 was isolated and shown to have the same phenotypic effects as when the gene is present on a 2μm plasmid. Genetic and Western blotting experiments indicated that Pdr13p exerts its effect on Pdr1p at a posttranslational step. These data support the view that Pdr13p influences pleiotropic drug resistance by enhancing the function of the transcriptional regulatory protein Pdr1p.

scholarly journals Transcriptional Activation in Yeast Cells Lacking Transcription Factor IIA

Genetics ◽  
1999 ◽  
Vol 153 (4) ◽  
pp. 1573-1581 ◽  
Author(s):  
Susanna Chou ◽  
Sukalyan Chatterjee ◽  
Mark Lee ◽  
Kevin Struhl
Keyword(s):  
Transcription Factor ◽  
Transcriptional Activation ◽  
Large Subunit ◽  
Yeast Cells ◽  
Specific Cell ◽  
Wild Type ◽  
Activation Domains ◽  
Cycle Arrest ◽  
General Transcription Factor

Abstract The general transcription factor IIA (TFIIA) forms a complex with TFIID at the TATA promoter element, and it inhibits the function of several negative regulators of the TATA-binding protein (TBP) subunit of TFIID. Biochemical experiments suggest that TFIIA is important in the response to transcriptional activators because activation domains can interact with TFIIA, increase recruitment of TFIID and TFIIA to the promoter, and promote isomerization of the TFIID-TFIIA-TATA complex. Here, we describe a double-shut-off approach to deplete yeast cells of Toa1, the large subunit of TFIIA, to <1% of the wild-type level. Interestingly, such TFIIA-depleted cells are essentially unaffected for activation by heat shock factor, Ace1, and Gal4-VP16. However, depletion of TFIIA causes a general two- to threefold decrease of transcription from most yeast promoters and a specific cell-cycle arrest at the G2-M boundary. These results indicate that transcriptional activation in vivo can occur in the absence of TFIIA.

scholarly journals CaNdt80 Is Involved in Drug Resistance in Candida albicans by Regulating CDR1

2004 ◽  
Vol 48 (12) ◽  
pp. 4505-4512 ◽  
Author(s):  
Chia-Geun Chen ◽  
Yun-Liang Yang ◽  
Hsin-I Shih ◽  
Chia-Li Su ◽  
Hsiu-Jung Lo
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Drug Resistance ◽  
Candida Albicans ◽  
Type Strain ◽  
Antifungal Agents ◽  
Wild Type Strain ◽  
Efflux Pump ◽  
Antifungal Drugs ◽  
Galactosidase Activity ◽  
Wild Type

ABSTRACT Overexpression of CDR1, an efflux pump, is one of the major mechanisms contributing to drug resistance in Candida albicans. CDR1 p-lacZ was constructed and transformed into a Saccharomyces cerevisiae strain so that the lacZ gene could be used as the reporter to monitor the activity of the CDR1 promoter. Overexpression of CaNDT80, the C. albicans homolog of S. cerevisiae NDT80, increases the β-galactosidase activity of the CDR1 p-lacZ construct in S. cerevisiae. Furthermore, mutations in CaNDT80 abolish the induction of CDR1 expression by antifungal agents in C. albicans. Consistently, the Candt80/Candt80 mutant is also more susceptible to antifungal drugs than the wild-type strain. Thus, the gene for CaNdt80 may be the first gene among the regulatory factors involved in drug resistance in C. albicans whose function has been identified.

The general transcription factor RAP30 binds to RNA polymerase II and prevents it from binding nonspecifically to DNA

1992 ◽  
Vol 12 (1) ◽  
pp. 30-37
Author(s):  
M T Killeen ◽  
J F Greenblatt
Keyword(s):  
Transcription Factor ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Small Subunit ◽  
Glutathione S Transferase ◽  
General Transcription Factor ◽  
Indirect Consequence ◽  
Transcription In Vitro ◽  
Sigma 70

RAP30/74 is a human general transcription factor that binds to RNA polymerase II and is required for initiation of transcription in vitro regardless of whether the promoter has a recognizable TATA box (Z. F. Burton, M. Killeen, M. Sopta, L. G. Ortolan, and J. F. Greenblatt, Mol. Cell. Biol. 8:1602-1613, 1988). Part of the amino acid sequence of RAP30, the small subunit of RAP30/74, has limited homology with part of Escherichia coli sigma 70 (M. Sopta, Z. F. Burton, and J. Greenblatt, Nature (London) 341:410-414, 1989). To determine which sigmalike activities of RAP30/74 could be attributed to RAP30, we purified human RAP30 and a RAP30-glutathione-S-transferase fusion protein that had been produced in E. coli. Bacterially produced RAP30 bound to RNA polymerase II in the absence of RAP74. Both partially purified natural RAP30/74 and recombinant RAP30 prevented RNA polymerase II from binding nonspecifically to DNA. In addition, nonspecific transcription by RNA polymerase II was greatly inhibited by RAP30-glutathione-S-transferase. DNA-bound RNA polymerase II could be removed from DNA by partially purified RAP30/74 but not by bacterially expressed RAP30. Thus, the ability of RAP30/74 to recruit RNA polymerase II to a promoter-bound preinitiation complex may be an indirect consequence of its ability to suppress nonspecific binding of RNA polymerase II to DNA.

Pleiotropic drug-resistance attenuated genomic library improves elucidation of drug mechanisms

2015 ◽  
Vol 11 (11) ◽  
pp. 3129-3136 ◽  
Author(s):  
Namal V. C. Coorey ◽  
James H. Matthews ◽  
David S. Bellows ◽  
Paul H. Atkinson
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Drug Resistance ◽  
Mechanism Of Action ◽  
Gene Deletion ◽  
Genomic Library ◽  
Deletion Mutants ◽  
Pleiotropic Drug Resistance ◽  
Genome Wide

Identifying Saccharomyces cerevisiae genome-wide gene deletion mutants that confer hypersensitivity to a xenobiotic aids the elucidation of its mechanism of action (MoA).

scholarly journals New Insights into the Pleiotropic Drug Resistance Network from Genome-Wide Characterization of the YRR1 Transcription Factor Regulation System

2002 ◽  
Vol 22 (8) ◽  
pp. 2642-2649 ◽  
Author(s):  
Stéphane Le Crom ◽  
Frédéric Devaux ◽  
Philippe Marc ◽  
Xiaoting Zhang ◽  
W. Scott Moye-Rowley ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Transcription Factor ◽  
Binding Site ◽  
Time Course ◽  
Target Genes ◽  
Regulation System ◽  
Dependent Manner ◽  
Pleiotropic Drug Resistance ◽  
Genome Wide

ABSTRACT Yrr1p is a recently described Zn2Cys6 transcription factor involved in the pleiotropic drug resistance (PDR) phenomenon. It is controlled in a Pdr1p-dependent manner and is autoregulated. We describe here a new genome-wide approach to characterization of the set of genes directly regulated by Yrr1p. We found that the time-course production of an artificial chimera protein containing the DNA-binding domain of Yrr1p activated the 15 genes that are also up-regulated by a gain-of-function mutant of Yrr1p. Gel mobility shift assays showed that the promoters of the genes AZR1, FLR1, SNG1, YLL056C, YLR346C, and YPL088W interacted with Yrr1p. The putative consensus Yrr1p binding site deduced from these experiments, (T/A)CCG(C/T)(G/T)(G/T)(A/T)(A/T), is strikingly similar to the PDR element binding site sequence recognized by Pdr1p and Pdr3p. The minor differences between these sequences are consistent with Yrr1p and Pdr1p and Pdr3p having different sets of target genes. According to these data, some target genes are directly regulated by Pdr1p and Pdr3p or by Yrr1p, whereas some genes are indirectly regulated by the activation of Yrr1p. Some genes, such as YOR1, SNQ2, and FLR1, are clearly directly controlled by both classes of transcription factor, suggesting an important role for the corresponding membrane proteins.

scholarly journals A Nuclear Import Pathway for a Protein Involved in tRNA Maturation

1997 ◽  
Vol 139 (7) ◽  
pp. 1655-1661 ◽  
Author(s):  
Jonathan S. Rosenblum ◽  
Lucy F. Pemberton ◽  
Günter Blobel
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Nuclear Import ◽  
Ribosomal Proteins ◽  
Ribosome Biogenesis ◽  
Sequence Similarity ◽  
Wild Type ◽  
Major Pathway ◽  
Trna Processing ◽  
Transport Factors ◽  
Limited Sequence

A limited number of transport factors, or karyopherins, ferry particular substrates between the cytoplasm and nucleoplasm. We identified the Saccharomyces cerevisiae gene YDR395w/SXM1 as a potential karyopherin on the basis of limited sequence similarity to known karyopherins. From yeast cytosol, we isolated Sxm1p in complex with several potential import substrates. These substrates included Lhp1p, the yeast homologue of the human autoantigen La that has recently been shown to facilitate maturation of pre-tRNA, and three distinct ribosomal proteins, Rpl16p, Rpl25p, and Rpl34p. Further, we demonstrate that Lhp1p is specifically imported by Sxm1p. In the absence of Sxm1p, Lhp1p was mislocalized to the cytoplasm. Sxm1p and Lhp1p represent the karyopherin and a cognate substrate of a unique nuclear import pathway, one that operates upstream of a major pathway of pre-tRNA maturation, which itself is upstream of tRNA export in wild-type cells. In addition, through its association with ribosomal proteins, Sxm1p may have a role in coordinating ribosome biogenesis with tRNA processing.

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