Role of SSD1 in Phenotypic Variation of Saccharomyces cerevisiae Strains Lacking DEG1-Dependent Pseudouridylation

Yeast phenotypes associated with the lack of wobble uridine (U34) modifications in tRNA were shown to be modulated by an allelic variation of SSD1, a gene encoding an mRNA-binding protein. We demonstrate that phenotypes caused by the loss of Deg1-dependent tRNA pseudouridylation are similarly affected by SSD1 allelic status. Temperature sensitivity and protein aggregation are elevated in deg1 mutants and further increased in the presence of the ssd1-d allele, which encodes a truncated form of Ssd1. In addition, chronological lifespan is reduced in a deg1 ssd1-d mutant, and the negative genetic interactions of the U34 modifier genes ELP3 and URM1 with DEG1 are aggravated by ssd1-d. A loss of function mutation in SSD1, ELP3, and DEG1 induces pleiotropic and overlapping phenotypes, including sensitivity against target of rapamycin (TOR) inhibitor drug and cell wall stress by calcofluor white. Additivity in ssd1 deg1 double mutant phenotypes suggests independent roles of Ssd1 and tRNA modifications in TOR signaling and cell wall integrity. However, other tRNA modification defects cause growth and drug sensitivity phenotypes, which are not further intensified in tandem with ssd1-d. Thus, we observed a modification-specific rather than general effect of SSD1 status on phenotypic variation in tRNA modification mutants. Our results highlight how the cellular consequences of tRNA modification loss can be influenced by protein targeting specific mRNAs.

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Saccharomyces cerevisiae Mid2p Is a Potential Cell Wall Stress Sensor and Upstream Activator of thePKC1-MPK1 Cell Integrity Pathway

Journal of Bacteriology ◽

10.1128/jb.181.11.3330-3340.1999 ◽

1999 ◽

Vol 181 (11) ◽

pp. 3330-3340 ◽

Cited By ~ 194

Author(s):

Troy Ketela ◽

Robin Green ◽

Howard Bussey

Keyword(s):

Saccharomyces Cerevisiae ◽

Cell Wall ◽

Wall Stress ◽

Structural Features ◽

Mitogen Activated Protein Kinase ◽

Cell Integrity ◽

Chitin Synthesis ◽

Calcofluor White ◽

Reading Frame ◽

Cell Wall Stress

ABSTRACT The MID2 gene of Saccharomyces cerevisiaeencodes a protein with structural features indicative of a plasma membrane-associated cell wall sensor. MID2 was isolated as a multicopy activator of the Skn7p transcription factor. Deletion ofMID2 causes resistance to calcofluor white, diminished production of stress-induced cell wall chitin under a variety of conditions, and changes in growth rate and viability in a number of different cell wall biosynthesis mutants. Overexpression ofMID2 causes hyperaccumulation of chitin and increased sensitivity to calcofluor white. α-Factor hypersensitivity ofmid2Δ mutants can be suppressed by overexpression of upstream elements of the cell integrity pathway, includingPKC1, RHO1, WSC1, andWSC2. Mid2p and Wsc1p appear to have overlapping roles in maintaining cell integrity since mid2Δ wsc1Δ mutants are inviable on medium that does not contain osmotic support. A role for MID2 in the cell integrity pathway is further supported by the finding that MID2 is required for induction of Mpk1p tyrosine phosphorylation during exposure to α-factor, calcofluor white, or high temperature. Our data are consistent with a role for Mid2p in sensing cell wall stress and in activation of a response that includes both increased chitin synthesis and the Mpk1p mitogen-activated protein kinase cell integrity pathway. In addition, we have identified an open reading frame, MTL1, which encodes a protein with both structural and functional similarity to Mid2p.

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Deletion of YLR358C Contributes to Reduce Cell Wall Integrity in Saccharomyces Cerevisiae

10.21203/rs.3.rs-1235133/v1 ◽

2022 ◽

Author(s):

Yu Zhang ◽

Mengyan Li ◽

Hanying Wang ◽

Juqing Deng ◽

Jianxing Liu ◽

...

Keyword(s):

Saccharomyces Cerevisiae ◽

Cell Wall ◽

Sodium Dodecyl ◽

Wall Stress ◽

Pathogenic Fungi ◽

Cell Wall Integrity ◽

Calcofluor White ◽

Fungal Cell ◽

Reading Frame ◽

Stress Signals

Abstract The mechanism of fungal cell wall synthesis and assembly is still unclear. Saccharomyces cerevisiae (S. cerevisiae) and pathogenic fungi are conserved in cell wall construction and response to stress signals, and often respond to cell wall stress through activated cell wall integrity (CWI) pathways. Whether the YLR358C open reading frame regulates CWI remains unclear. This study found that the growth of S. cerevisiae with YLR358C knockout was significantly inhibited on the medium containing different concentrations of cell wall interfering agents Calcofluor White (CFW), Congo Red (CR) and sodium dodecyl sulfate (SDS). CFW staining showed that the cell wall chitin was down-regulated, and transmission electron microscopy also observed a decrease in cell wall thickness. Transcriptome sequencing and analysis showed that YLR358C gene may be involved in the regulation of CWI signaling pathway. It was found by qRT-PCR that WSC3, SWI4 and HSP12 were differentially expressed after YLR358C was knocked out. The above results suggest that YLR358C may regulate the integrity of the yeast cell walls and has some potential for application in fermentation.

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Cbk1 kinase and Bck2 control MAP kinase activation and inactivation during heat shock

Molecular Biology of the Cell ◽

10.1091/mbc.e11-04-0371 ◽

2011 ◽

Vol 22 (24) ◽

pp. 4892-4907 ◽

Cited By ~ 15

Author(s):

Venkata K. Kuravi ◽

Cornelia Kurischko ◽

Manasi Puri ◽

Francis C. Luca

Keyword(s):

Gene Expression ◽

Cell Wall ◽

Heat Shock ◽

Wall Stress ◽

Mitogen Activated Protein Kinase ◽

Stress Signaling ◽

Cell Integrity ◽

Loss Of Function ◽

Kinase Activation ◽

Cell Wall Stress

Saccharomyces cerevisiae Cbk1 kinase is a LATS/NDR tumor suppressor orthologue and component of the Regulation of Ace2 and Morphogenesis signaling network. Cbk1 was previously implicated in regulating polarized morphogenesis, gene expression, and cell integrity. Here we establish that Cbk1 is critical for heat shock and cell wall stress signaling via Bck2, a protein associated with the Pkc1-Mpk1 cell integrity pathway. We demonstrate that cbk1 and bck2 loss-of-function mutations prevent Mpk1 kinase activation and Mpk1-dependent gene expression but do not disrupt Mpk1 Thr-190/Tyr-192 phosphorylation. Bck2 overexpression partially restores Mpk1-dependent Rlm1 transcription factor activity in cbk1 mutants, suggesting that Bck2 functions downstream of Cbk1. We demonstrate that Bck2 precisely colocalizes with the mitogen-activated protein kinase (MAPK) phosphatase Sdp1. During heat shock, Bck2 and Sdp1 transiently redistribute from nuclei and the cytosol to mitochondria and other cytoplasmic puncta before returning to their pre-stressed localization patterns. Significantly, Cbk1 inhibition delays the return of Bck2 and Sdp1 to their pre-stressed localization patterns and delays Mpk1 Thr-190/Tyr-192 dephosphorylation upon heat shock adaptation. We conclude that Cbk1 and Bck2 are required for Mpk1 activation during heat shock and cell wall stress and for Mpk1 dephosphorylation during heat shock adaptation. These data provide the first evidence that Cbk1 kinase regulates MAPK-dependent stress signaling and provide mechanistic insight into Sdp1 phosphatase regulation.

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Emw1p/YNL313cp is essential for maintenance of the cell wall in Saccharomyces cerevisiae

Microbiology ◽

10.1099/mic.0.045971-0 ◽

2011 ◽

Vol 157 (4) ◽

pp. 1032-1041 ◽

Cited By ~ 3

Author(s):

Tatjana Sipling ◽

Chao Zhai ◽

Barry Panaretou

Keyword(s):

Saccharomyces Cerevisiae ◽

Cell Wall ◽

Permissive Temperature ◽

Loss Of Function ◽

Chitin Synthesis ◽

Calcofluor White ◽

Compensatory Response ◽

Protein Protein Interaction ◽

Kinase Cascade ◽

A Cell

There are six essential genes in the Saccharomyces cerevisiae genome which encode proteins bearing the tetratricopeptide repeat (TPR) domain that mediates protein–protein interaction. Thus far, the function of one of them, YNL313c, remains unknown. Our conditional mutants of YNL313c display osmoremedial temperature sensitivity, hypersensitivity to both Calcofluor White and low concentrations of SDS, and osmoremedial caffeine sensitivity. These are hallmarks of mutants that display cell wall defects. Accordingly we rename the gene as EMW1 (essential for maintenance of the cell wall). Loss of Emw1p function is not associated with abrogation of the cell wall integrity (CWI) MAP kinase cascade. Instead, emw1ts mutants activate this cascade even at permissive temperature, indicating that loss of Emw1p function does not cause a defect in sensors and effectors of cell wall signalling, but leads to a cell wall defect directly. Constitutive activation of the CWI cascade is reflected by the overproduction of chitin by emw1ts mutants, a compensatory response frequently displayed by cell wall mutants. Growth is restored to emw1ts mutants incubated at otherwise non-permissive temperature when GFA1 is overexpressed. GFA1 encodes the hexosephosphate aminotransferase that catalyses the rate-limiting step in the pathway that synthesizes the chitin precursor UDP-GlcNAc. The possibility that Emw1p is required for function of Gfa1p was ruled out, because the emw1ts phenotype persists when the requirement for Gfa1p is bypassed. Furthermore, if loss of Emw1p function leads to loss of function of Gfa1p, then chitin synthesis would be diminished. Instead, a stimulation of the synthesis of this polymer is detected. Consequently, the defect associated with emw1ts mutants may be associated with compromise in one of the remaining processes that depend on UDP-GlcNAc, namely N-glycosylation or glycosylphosphatidylinositol (GPI)-anchor synthesis.

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Stepwise Decrease in Daptomycin Susceptibility in Clinical Staphylococcus aureus Isolates Associated with an Initial Mutation inrpoBand a Compensatory Inactivation of theclpXGene

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01303-15 ◽

2015 ◽

Vol 59 (11) ◽

pp. 6983-6991 ◽

Cited By ~ 29

Author(s):

Kristoffer T. Bæk ◽

Louise Thøgersen ◽

René G. Mogenssen ◽

Maiken Mellergaard ◽

Line E. Thomsen ◽

...

Keyword(s):

Staphylococcus Aureus ◽

Cell Wall ◽

Wall Stress ◽

Growth Defect ◽

Loss Of Function ◽

Genetic Changes ◽

Content Type ◽

Virulence Traits ◽

Lipopeptide Antibiotic

ABSTRACTDaptomycin is a lipopeptide antibiotic used clinically for the treatment of methicillin-resistantStaphylococcus aureus(MRSA) infections. The emergence of daptomycin-nonsusceptibleS. aureusisolates during therapy is often associated with multiple genetic changes; however, the relative contributions of these changes to resistance and other phenotypic changes usually remain unclear. The present study was undertaken to investigate this issue using a genetically characterized series of four isogenic clinical MRSA strains derived from a patient with bacteremia before and during daptomycin treatment. The first strain obtained after daptomycin therapy carried a single-nucleotide polymorphism (SNP) inrpoB(RpoB A477D) that decreased susceptibility not only to daptomycin but also to vancomycin, β-lactams, and rifampin. Furthermore, therpoBmutant exhibited pleiotropic phenotypes, including increased cell wall thickness, reduced expression of virulence traits, induced expression of the stress-associated transcriptional regulator Spx, and slow growth. A subsequently acquired loss-of-function mutation inclpXpartly alleviated the growth defect conferred by therpoBmutation without changing antibiotic susceptibility. The final isolate acquired three additional mutations, including an SNP inmprF(MprF S295L) known to confer daptomycin nonsusceptibility, and accordingly, this isolate was the only daptomycin-nonsusceptible strain of this series. Interestingly, in this isolate, the cell wall had regained the same thickness as that of the parental strain, while the level of transcription of thevraSR(cell wall stress regulator) was increased. In conclusion, this study illustrates how serial genetic changes selectedin vivocontribute to daptomycin nonsusceptibility, growth fitness, and virulence traits.

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Faculty Opinions recommendation of Transcriptional profiling reveals that daptomycin induces the Staphylococcus aureus cell wall stress stimulon and genes responsive to membrane depolarization.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1098746.558329 ◽

2008 ◽

Author(s):

Lynn Silver

Keyword(s):

Staphylococcus Aureus ◽

Cell Wall ◽

Transcriptional Profiling ◽

Wall Stress ◽

Membrane Depolarization ◽

Cell Wall Stress

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The Regulatory Function of the Molecular Chaperone Hsp90 in the Cell Wall Integrity of Pathogenic Fungi

Current Proteomics ◽

10.2174/1570164615666180820155807 ◽

2018 ◽

Vol 16 (1) ◽

pp. 44-53

Author(s):

Marina Campos Rocha ◽

Camilla Alves Santos ◽

Iran Malavazi

Keyword(s):

Cell Wall ◽

Antifungal Therapy ◽

Molecular Chaperone ◽

Regulatory Protein ◽

Pathogenic Fungi ◽

Antifungal Drugs ◽

Cell Wall Integrity ◽

Regulatory Function ◽

Loss Of Function ◽

Hsp90 Inhibition

Different signaling cascades including the Cell Wall Integrity (CWI), the High Osmolarity Glycerol (HOG) and the Ca2+/calcineurin pathways control the cell wall biosynthesis and remodeling in fungi. Pathogenic fungi, such as Aspergillus fumigatus and Candida albicans, greatly rely on these signaling circuits to cope with different sources of stress, including the cell wall stress evoked by antifungal drugs and the host’s response during infection. Hsp90 has been proposed as an important regulatory protein and an attractive target for antifungal therapy since it stabilizes major effector proteins that act in the CWI, HOG and Ca2+/calcineurin pathways. Data from the human pathogen C. albicans have provided solid evidence that loss-of-function of Hsp90 impairs the evolution of resistance to azoles and echinocandin drugs. In A. fumigatus, Hsp90 is also required for cell wall integrity maintenance, reinforcing a coordinated function of the CWI pathway and this essential molecular chaperone. In this review, we focus on the current information about how Hsp90 impacts the aforementioned signaling pathways and consequently the homeostasis and maintenance of the cell wall, highlighting this cellular event as a key mechanism underlying antifungal therapy based on Hsp90 inhibition.

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In vitro Cell Wall Stress Assay for Fusarium oxysporum

BIO-PROTOCOL ◽

10.21769/bioprotoc.1915 ◽

2016 ◽

Vol 6 (17) ◽

Cited By ~ 1

Author(s):

Elena Pérez-Nadales ◽

Antonio Di Pietro

Keyword(s):

Cell Wall ◽

Fusarium Oxysporum ◽

Wall Stress ◽

Cell Wall Stress

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Yeast Nap1-Binding Protein Nbp2p Is Required for Mitotic Growth at High Temperatures and for Cell Wall Integrity

Genetics ◽

10.1093/genetics/165.2.517 ◽

2003 ◽

Vol 165 (2) ◽

pp. 517-529

Author(s):

Kentaro Ohkuni ◽

Asuko Okuda ◽

Akihiko Kikuchi

Keyword(s):

Cell Death ◽

Cell Wall ◽

High Temperature ◽

High Temperatures ◽

Hybrid System ◽

Mapk Pathway ◽

Binding Protein ◽

Cell Wall Integrity ◽

Calcofluor White ◽

Two Hybrid

AbstractNbp2p is a Nap1-binding protein in Saccharomyces cerevisiae identified by its interaction with Nap1 by a two-hybrid system. NBP2 encodes a novel protein consisting of 236 amino acids with a Src homology 3 (SH3) domain. We showed that NBP2 functions to promote mitotic cell growth at high temperatures and cell wall integrity. Loss of Nbp2 results in cell death at high temperatures and in sensitivity to calcofluor white. Cell death at high temperature is thought not to be due to a weakened cell wall. Additionally, we have isolated several type-2C serine threonine protein phosphatases (PTCs) as multicopy suppressors and MAP kinase-kinase (MAPKK), related to the yeast PKC MAPK pathway, as deletion suppressors of the nbp2Δ mutant. Screening for deletion suppressors is a new genetic approach to identify and characterize additional proteins in the Nbp2-dependent pathway. Genetic analyses suggested that Ptc1, which interacts with Nbp2 by the two-hybrid system, acts downstream of Nbp2 and that cells lacking the function of Nbp2 prefer to lose Mkk1, but the PKC MAPK pathway itself is indispensable when Nbp2 is deleted at high temperature.

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