scholarly journals Cns1 Is an Essential Protein Associated with the Hsp90 Chaperone Complex in Saccharomyces cerevisiae That Can Restore Cyclophilin 40-Dependent Functions in cpr7ΔCells

1998 ◽  
Vol 18 (12) ◽  
pp. 7353-7359 ◽  
Author(s):  
James A. Marsh ◽  
Helen M. Kalton ◽  
Richard F. Gaber
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Molecular Chaperone ◽  
Single Gene ◽  
Normal Growth ◽  
Maximal Activity ◽  
Negative Regulation ◽  
Growth Defect ◽  
Chaperone Complex ◽  
Cyclophilin 40

ABSTRACT Saccharomyces cerevisiae harbors two cyclophilin 40-type enzymes, Cpr6 and Cpr7, which are components of the Hsp90 molecular chaperone machinery. Cpr7 is required for normal growth and is required for maximal activity of heterologous Hsp90-dependent substrates, including glucocorticoid receptor (GR) and the oncogenic tyrosine kinase pp60v-src . In addition, it has recently been shown that Cpr7 plays a major role in negative regulation of the S. cerevisiae heat shock transcription factor (HSF). To better understand functions associated with Cpr7, a search was undertaken for multicopy suppressors of the cpr7Δ slow-growth phenotype. The screen identified a single gene, designatedCNS1 (for cyclophilin seven suppressor), capable of suppressing the cpr7Δ growth defect. Overexpression ofCNS1 in cpr7Δ cells also largely restored GR activity and negative regulation of HSF. In vitro protein retention experiments in which Hsp90 heterocomplexes were precipitated resulted in coprecipitation of Cns1. Interaction between Cns1 and the carboxy terminus of Hsp90 was also shown by two-hybrid analysis. The functional consequences of CNS1 overexpression and its physical association with the Hsp90 machinery indicate that Cns1 is a previously unidentified component of molecular chaperone complexes. Thus far, Cns1 is the only tetratricopeptide repeat-containing component of Hsp90 heterocomplexes found to be essential for cell viability under all conditions tested.

scholarly journals Genomic Analysis Identifies NovelPseudomonas aeruginosaResistance Genes under Selection during Inhaled Aztreonam TherapyIn Vivo

2019 ◽  
Vol 63 (9) ◽  
Author(s):  
Kathryn McLean ◽  
Duankun Lee ◽  
Elizabeth A. Holmes ◽  
Kelsi Penewit ◽  
Adam Waalkes ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Positive Selection ◽  
Single Gene ◽  
Genomic Analysis ◽  
Maximal Activity ◽  
Content Type ◽  
Cystic Fibrosis Airway ◽  
Bacterial Fitness

ABSTRACTInhaled aztreonam is increasingly used for chronicPseudomonas aeruginosasuppression in patients with cystic fibrosis (CF), but the potential for that organism to evolve aztreonam resistance remains incompletely explored. Here, we performed genomic analysis of clonally related pre- and posttreatment CF clinical isolate pairs to identify genes that are under positive selection during aztreonam therapyin vivo. We identified 16 frequently mutated genes associated with aztreonam resistance, the most prevalent beingftsIandampC, and 13 of which increased aztreonam resistance when introduced as single gene transposon mutants. Several previously implicated aztreonam resistance genes were found to be under positive selection in clinical isolates even in the absence of inhaled aztreonam exposure, indicating that other selective pressures in the cystic fibrosis airway can promote aztreonam resistance. Given its potential to confer plasmid-mediated resistance, we further characterized mutantampCalleles and performed artificial evolution ofampCfor maximal activity against aztreonam. We found that naturally occurringampCmutants conferred variably increased resistance to aztreonam (2- to 64-fold) and other β-lactam agents but that its maximal evolutionary capacity for hydrolyzing aztreonam was considerably higher (512- to 1,024-fold increases) and was achieved while maintaining or increasing resistance to other drugs. These studies implicate novel chromosomal aztreonam resistance determinants while highlighting that different mutations are favored during selectionin vivoandin vitro, show thatampChas a high maximal potential to hydrolyze aztreonam, and provide an approach to disambiguate mutations promoting specific resistance phenotypes from those more generally increasing bacterial fitnessin vivo.

scholarly journals Alteration of the Protein Kinase Binding Domain Enhances Function of the Saccharomyces cerevisiae Molecular Chaperone Cdc37

2007 ◽  
Vol 6 (8) ◽  
pp. 1363-1372 ◽  
Author(s):  
Min Ren ◽  
Arti Santhanam ◽  
Paul Lee ◽  
Avrom Caplan ◽  
Stephen Garrett
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Protein Kinase ◽  
Protein Kinases ◽  
Molecular Chaperone ◽  
Binding Domain ◽  
Growth Defect ◽  
Temperature Sensitive ◽  
Physical Interaction ◽  
General Function ◽  
Amino Terminal

ABSTRACT Cdc37 is a molecular chaperone that has a general function in the biogenesis of protein kinases. We identified mutations within the putative “protein kinase binding domain” of Cdc37 that alleviate the conditional growth defect of a strain containing a temperature-sensitive allele, tpk2(Ts), of the cyclic AMP-dependent protein kinase (PKA). These dominant mutations alleviate the temperature-sensitive growth defect by elevating PKA activity, as judged by their effects on PKA-regulated processes, localization and phosphorylation of the PKA effector Msn2, as well as in vitro PKA activity. Although the tpk2(Ts) growth defect is also alleviated by Cdc37 overproduction, the CDC37 dominant mutants contain wild-type Cdc37 protein levels. In addition, Saccharomyces cerevisiae Ste11 protein kinase has an elevated physical interaction with the altered Cdc37 protein. These results implicate specific amino-terminal residues in the interaction between Cdc37 and client protein kinases and provide further genetic and biochemical support for a model in which Cdc37 functions as a molecular chaperone for protein kinases.

scholarly journals Loss-of-Function Mutations in HspR Rescue the Growth Defect of a Mycobacterium tuberculosis Proteasome Accessory Factor E (pafE) Mutant

2017 ◽  
Vol 199 (7) ◽  
Author(s):  
Jordan B. Jastrab ◽  
Marie I. Samanovic ◽  
Richard Copin ◽  
Bo Shopsin ◽  
K. Heran Darwin
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Heat Shock ◽  
Protein Quality ◽  
Normal Growth ◽  
Culture Conditions ◽  
Growth Defect ◽  
Loss Of Function ◽  
Content Type ◽  
Accessory Factor

ABSTRACT Mycobacterium tuberculosis uses a proteasome to degrade proteins by both ATP-dependent and -independent pathways. While much has been learned about ATP-dependent degradation, relatively little is understood about the ATP-independent pathway, which is controlled by Mycobacterium tuberculosis proteasome accessory factor E (PafE). Recently, we found that a Mycobacterium tuberculosis pafE mutant has slowed growth in vitro and is sensitive to killing by heat stress. However, we did not know if these phenotypes were caused by an inability to degrade the PafE-proteasome substrate HspR (heat shock protein repressor), an inability to degrade any damaged or misfolded proteins, or a defect in another protein quality control pathway. To address this question, we characterized pafE suppressor mutants that grew similarly to pafE + bacteria under normal culture conditions. All but one suppressor mutant analyzed contained mutations that inactivated HspR function, demonstrating that the slowed growth and heat shock sensitivity of a pafE mutant were caused primarily by the inability of the proteasome to degrade HspR. IMPORTANCE Mycobacterium tuberculosis encodes a proteasome that is highly similar to eukaryotic proteasomes and is required for virulence. We recently discovered a proteasome cofactor, PafE, which is required for the normal growth, heat shock resistance, and full virulence of M. tuberculosis. In this study, we demonstrate that PafE influences this phenotype primarily by promoting the expression of protein chaperone genes that are necessary for surviving proteotoxic stress.

scholarly journals Phosphorylation and Maximal Activity of Saccharomyces cerevisiae Meiosis-Specific Transcription Factor Ndt80 Is Dependent on Ime2

2002 ◽  
Vol 22 (20) ◽  
pp. 7024-7040 ◽  
Author(s):  
Richelle Sopko ◽  
Sheetal Raithatha ◽  
David Stuart
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Transcription Factor ◽  
Target Genes ◽  
Meiotic Chromosome ◽  
Maximal Activity ◽  
Direct Role ◽  
Specific Transcription Factor ◽  
Mutant Strains ◽  
Sporulation Genes

ABSTRACT The Saccharomyces cerevisiae meiosis-specific transcription factor Ndt80 is responsible for the induction of a class of genes referred to as middle sporulation genes. Among the members of this family are the B-type cyclins and other genes whose products are required for meiotic chromosome division and spore morphogenesis. Inactivation of NDT80 leads to a failure to induce the middle sporulation genes and a subsequent arrest in pachytene. The expression of NDT80 is itself highly regulated. The initial transcription of NDT80 is dependent upon the protein kinase Ime2; once Ndt80 protein accumulates, it activates its own promoter, thus generating an autoactivation loop. In addition to being transcriptionally regulated, Ndt80 protein is posttranslationally regulated. Phosphorylation of Ndt80 occurs coincident with its activation as a transcription factor. If expressed prematurely in meiosis, Ndt80 accumulates initially in an unmodified form that is subsequently modified by phosphorylation. In contrast, Ndt80 expressed in ime2 mutant strains does not become modified and has a reduced ability to activate transcription of its target genes. Ime2 can also phosphorylate Ndt80 in vitro, further supporting a direct role for Ime2 in the phosphorylation of Ndt80. These data indicate that Ime2 plays a novel and previously unexpected role in promoting chromosome dissemination and progress through meiotic development by activating Ndt80.

scholarly journals Genomewide Screening for Genes Associated with Gliotoxin Resistance and Sensitivity in Saccharomyces cerevisiae

2008 ◽  
Vol 52 (4) ◽  
pp. 1325-1329 ◽  
Author(s):  
Georgios Chamilos ◽  
Russell E. Lewis ◽  
Gregory A. Lamaris ◽  
Nathaniel D. Albert ◽  
Dimitrios P. Kontoyiannis
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Mitochondrial Function ◽  
Unknown Function ◽  
Single Gene ◽  
Normal Growth ◽  
Wild Type ◽  
Fourfold Increase ◽  
Genes Encoding ◽  
Increased Sensitivity ◽  
Secondary Fungal Metabolite

ABSTRACT Gliotoxin (GT) is a secondary fungal metabolite with pleiotropic immunosuppressive properties that have been implicated in Aspergillus virulence. However, the mechanisms of GT cytotoxicity and its molecular targets in eukaryotic cells have not been fully characterized. We screened a haploid library of Saccharomyces cerevisiae single-gene deletion mutants (4,787 strains in EUROSCARF) to identify nonessential genes associated with GT increased resistance (GT-IR) and increased sensitivity (GT-IS). The susceptibility of the wild-type parental strain BY4741 to GT was initially assessed by broth microdilution methods using different media. GT-IR and GT-IS were defined as a fourfold increase and decrease, respectively, in MIC, and this was additionally confirmed by susceptibility testing on agar yeast extract-peptone-glucose plates. The specificity of GT-IR and GT-IS mutants exhibiting normal growth compared with the wild-type strain was further tested in studies of their susceptibility to conventional antifungal agents, cycloheximide, and H2O2. GT-IR was associated with the disruption of genes acting in general metabolism (OPI1, SNF1, IFA38), mitochondrial function (RTG2), DNA damage repair (RAD18), and vesicular transport (APL2) and genes of unknown function (YGL235W, YOR345C, YLR456W, YGL072C). The disruption of three genes encoding transsulfuration (CYS3), mitochondrial function (MEF2), and an unknown function (YKL037W) led to GT-IS. Specificity for GT-IR and GT-IS was observed in all mutants. Importantly, the majority (69%) of genes implicated in GT-IR (6/10) and GT-IS (2/3) have human homologs. We identified novel Saccharomyces genes specifically implicated in GT-IR or GT-IS. Because most of these genes are evolutionarily conserved, further characterization of their function could improve our understanding of GT cytotoxicity mechanisms in humans.

scholarly journals Wss1 Is a SUMO-Dependent Isopeptidase That Interacts Genetically with the Slx5-Slx8 SUMO-Targeted Ubiquitin Ligase

2010 ◽  
Vol 30 (15) ◽  
pp. 3737-3748 ◽  
Author(s):  
Janet R. Mullen ◽  
Chi-Fu Chen ◽  
Steven J. Brill
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Fusion Protein ◽  
Ubiquitin Ligase ◽  
Copy Number ◽  
Dna Helicase ◽  
Genome Integrity ◽  
Growth Defect ◽  
Sumo Fusion ◽  
Isopeptidase Activity

ABSTRACT Protein sumoylation plays an important but poorly understood role in controlling genome integrity. In Saccharomyces cerevisiae, the Slx5-Slx8 SUMO-targeted Ub ligase appears to be needed to ubiquitinate sumoylated proteins that arise in the absence of the Sgs1 DNA helicase. WSS1, a high-copy-number suppressor of a mutant SUMO, was implicated in this pathway because it shares phenotypes with SLX5-SLX8 mutants, including a wss1Δ sgs1Δ synthetic-fitness defect. Here we show that Wss1, a putative metalloprotease, physically binds SUMO and displays in vitro isopeptidase activity on poly-SUMO chains. Like that of SLX5, overexpression of WSS1 suppresses sgs1Δ slx5Δ lethality and the ulp1ts growth defect. Interestingly, although Wss1 is relatively inactive on ubiquitinated substrates and poly-Ub chains, it efficiently deubiquitinates a Ub-SUMO isopeptide conjugate and a Ub-SUMO fusion protein. Wss1 was further implicated in Ub metabolism on the basis of its physical association with proteasomal subunits. The results suggest that Wss1 is a SUMO-dependent isopeptidase that acts on sumoylated substrates as they undergo proteasomal degradation.

scholarly journals Bni5p, a Septin-Interacting Protein, Is Required for Normal Septin Function and Cytokinesis in Saccharomyces cerevisiae

2002 ◽  
Vol 22 (19) ◽  
pp. 6906-6920 ◽  
Author(s):  
Philip R. Lee ◽  
Sukgil Song ◽  
Hyeon-Su Ro ◽  
Chong J. Park ◽  
John Lippincott ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Protein Localization ◽  
Growth Defect ◽  
Dependent Manner ◽  
Interacting Protein ◽  
Growth Defects ◽  
Bud Emergence ◽  
Bud Neck ◽  
And Function

ABSTRACT In the budding yeast Saccharomyces cerevisiae, the Cdc3p, Cdc10p, Cdc11p, Cdc12p, and Sep7p/Shs1p septins assemble early in the cell cycle in a ring that marks the future cytokinetic site. The septins appear to be major structural components of a set of filaments at the mother-bud neck and function as a scaffold for recruiting proteins involved in cytokinesis and other processes. We isolated a novel gene, BNI5, as a dosage suppressor of the cdc12-6 growth defect. Overexpression of BNI5 also suppressed the growth defects of cdc10-1, cdc11-6, and sep7Δ strains. Loss of BNI5 resulted in a cytokinesis defect, as evidenced by the formation of connected cells with shared cytoplasms, and deletion of BNI5 in a cdc3-6, cdc10-1, cdc11-6, cdc12-6, or sep7Δ mutant strain resulted in enhanced defects in septin localization and cytokinesis. Bni5p localizes to the mother-bud neck in a septin-dependent manner shortly after bud emergence and disappears from the neck approximately 2 to 3 min before spindle disassembly. Two-hybrid, in vitro binding, and protein-localization studies suggest that Bni5p interacts with the N-terminal domain of Cdc11p, which also appears to be sufficient for the localization of Cdc11p, its interaction with other septins, and other critical aspects of its function. Our data suggest that the Bni5p-septin interaction is important for septin ring stability and function, which is in turn critical for normal cytokinesis.

scholarly journals Saccharomyces cerevisiae RAI1 (YGL246c) Is Homologous to Human DOM3Z and Encodes a Protein That Binds the Nuclear Exoribonuclease Rat1p

2000 ◽  
Vol 20 (11) ◽  
pp. 4006-4015 ◽  
Author(s):  
Yang Xue ◽  
Xinxue Bai ◽  
Insuk Lee ◽  
George Kallstrom ◽  
Jennifer Ho ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Genetic Interaction ◽  
Rna Degradation ◽  
Growth Defect ◽  
Nuclear Targeting ◽  
Interacting Protein ◽  
Rrna Species ◽  
Synthetically Lethal

ABSTRACT The RAT1 gene of Saccharomyces cerevisiaeencodes a 5′→3′ exoribonuclease which plays an essential role in yeast RNA degradation and/or processing in the nucleus. We have cloned a previously uncharacterized gene (YGL246c) that we refer to asRAI1 (Rat1p interacting protein 1). RAI1 is homologous to Caenorhabditis elegans DOM-3 and humanDOM3Z. Deletion of RAI1 confers a growth defect which can be complemented by an additional copy of RAT1 on a centromeric vector or by directing Xrn1p, the cytoplasmic homolog of Rat1p, to the nucleus through the addition of a nuclear targeting sequence. Deletion of RAI1 is synthetically lethal with therat1-1ts mutation and shows genetic interaction with a deletion of SKI2 but not XRN1. Polysome analysis of an rai1 deletion mutant indicated a defect in 60S biogenesis which was nearly fully reversed by high-copyRAT1. Northern blot analysis of rRNAs revealed thatrai1 is required for normal 5.8S processing. In the absence of RAI1, 5.8SL was the predominant form of 5.8S and there was an accumulation of 3′-extended forms but not 5′-extended species of 5.8S. In addition, a 27S pre-rRNA species accumulated in therai1 mutant. Thus, deletion of RAI1 affects both 5′ and 3′ processing reactions of 5.8S rRNA. Consistent with the in vivo data suggesting that RAI1 enhances RAT1function, purified Rai1p stabilized the in vitro exoribonuclease activity of Rat1p.

WdChs4p, a Homolog of Chitin Synthase 3 in Saccharomyces cerevisiae , Alone Cannot Support Growth of Wangiella ( Exophiala ) dermatitidis at the Temperature of Infection

1999 ◽  
Vol 67 (12) ◽  
pp. 6619-6630
Author(s):  
Zheng Wang ◽  
Li Zheng ◽  
Melinda Hauser ◽  
Jeffery M. Becker ◽  
Paul J. Szaniszlo
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Pathogenic Fungus ◽  
Maximal Activity ◽  
Northern Blotting ◽  
Significant Loss ◽  
Chitin Synthase ◽  
Wall Structure ◽  
Triple Mutant ◽  
Ph Optimum

ABSTRACT By using improved transformation methods for Wangiella dermatitidis , and a cloned fragment of its chitin synthase 4 structural gene ( WdCHS4 ) as a marking sequence, the full-length gene was rescued from the genome of this human pathogenic fungus. The encoded chitin synthase product (WdChs4p) showed high homology with Chs3p of Saccharomyces cerevisiae and other class IV chitin synthases, and Northern blotting showed that WdCHS4 was expressed at constitutive levels under all conditions tested. Reduced chitin content, abnormal yeast clumpiness and budding kinetics, and increased melanin secretion resulted from the disruption of WdCHS4 suggesting that WdChs4p influences cell wall structure, cellular reproduction, and melanin deposition, respectively. However, no significant loss of virulence was detected when the wdchs4Δ strain was tested in an acute mouse model. Using a wdchs1Δ wdchs2Δ wdchs3Δ triple mutant of W. dermatitidis , which grew poorly but adequately at 25°C, we assayed WdChs4p activity in the absence of activities contributed by its three other WdChs proteins. Maximal activity required trypsin activation, suggesting a zymogenic nature. The activity also had a pH optimum of 7.5, was most stimulated by Mg 2+ , and was more inhibited by polyoxin D than by nikkomycin Z. Although the WdChs4p activity had a broad temperature optimum between 30 to 45°C in vitro, this activity alone did not support the growth of the wdchs1Δ wdchs2Δ wdchs3Δ triple mutant at 37°C, a temperature commensurate with infection.

scholarly journals Heat Shock Element Architecture Is an Important Determinant in the Temperature and Transactivation Domain Requirements for Heat Shock Transcription Factor

1998 ◽  
Vol 18 (11) ◽  
pp. 6340-6352 ◽  
Author(s):  
Nicholas Santoro ◽  
Nina Johansson ◽  
Dennis J. Thiele
Keyword(s):  
Transcription Factor ◽  
Heat Shock ◽  
Transcriptional Activation ◽  
Single Gene ◽  
Limited Proteolysis ◽  
Normal Growth ◽  
Heat Shock Transcription Factor ◽  
Transactivation Domain ◽  
Heat Shock Element

ABSTRACT The baker’s yeast Saccharomyces cerevisiae possesses a single gene encoding heat shock transcription factor (HSF), which is required for the activation of genes that participate in stress protection as well as normal growth and viability. Yeast HSF (yHSF) contains two distinct transcriptional activation regions located at the amino and carboxyl termini. Activation of the yeast metallothionein gene, CUP1, depends on a nonconsensus heat shock element (HSE), occurs at higher temperatures than other heat shock-responsive genes, and is highly dependent on the carboxyl-terminal transactivation domain (CTA) of yHSF. The results described here show that the noncanonical (or gapped) spacing of GAA units in the CUP1HSE (HSE1) functions to limit the magnitude of CUP1transcriptional activation in response to heat and oxidative stress. The spacing in HSE1 modulates the dependence for transcriptional activation by both stresses on the yHSF CTA. Furthermore, a previously uncharacterized HSE in the CUP1 promoter, HSE2, modulates the magnitude of the transcriptional activation of CUP1, via HSE1, in response to stress. In vitro DNase I footprinting experiments suggest that the occupation of HSE2 by yHSF strongly influences the manner in which yHSF occupies HSE1. Limited proteolysis assays show that HSF adopts a distinct protease-sensitive conformation when bound to the CUP1HSE1, providing evidence that the HSE influences DNA-bound HSF conformation. Together, these results suggest that CUP1regulation is distinct from that of other classic heat shock genes through the interaction of yHSF with two nonconsensus HSEs. Consistent with this view, we have identified other gene targets of yHSF containing HSEs with sequence and spacing features similar to those ofCUP1 HSE1 and show a correlation between the spacing of the GAA units and the relative dependence on the yHSF CTA.

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