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Molecular and Cellular Biology Jan 2021Copper homeostasis is crucial for various cellular processes. The balance between nutritional and toxic copper levels is maintained through the regulation of its uptake,...
Copper homeostasis is crucial for various cellular processes. The balance between nutritional and toxic copper levels is maintained through the regulation of its uptake, distribution, and detoxification via antagonistic actions of two transcription factors, Ace1 and Mac1. Ace1 responds to toxic copper levels by transcriptionally regulating detoxification genes and Cup1 metallothionein confers protection against toxic copper levels. gene regulation is a multifactorial event requiring Ace1, TATA-binding protein (TBP), chromatin remodeler, acetyltransferase (Spt10), and histones. However, the role of histone H3 residues has not been fully elucidated. To investigate the role of the H3 tail in transcriptional regulation, we screened the library of histone mutants in copper stress. We identified mutations in H3 (K23Q, K27R, K36Q, Δ5-16, Δ13-16, Δ13-28, Δ25-28, Δ28-31, and Δ29-32) that reduce expression. We detected reduced Ace1 occupancy across the promoter in K23Q, K36Q, Δ5-16, Δ13-28, Δ25-28, and Δ28-31 mutations correlating with the reduced transcription. The majority of these mutations affect TBP occupancy at the promoter, augmenting the transcription defect. Additionally, some mutants displayed cytosolic protein aggregation upon copper stress. Altogether, our data establish previously unidentified residues of the H3 N-terminal tail and their modifications in regulation.
Topics: Amino Acid Sequence; Copper; DNA-Binding Proteins; Gene Expression Regulation, Fungal; Histone Acetyltransferases; Histones; Homeostasis; Metallothionein; Mutation; Nuclear Proteins; Promoter Regions, Genetic; Protein Binding; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Signal Transduction; Stress, Physiological; TATA-Box Binding Protein; Transcription Factors; Transcription, Genetic
PubMed: 33257505
DOI: 10.1128/MCB.00210-20 -
Molecular Biology of the Cell Jul 2021Sterols are important lipid components of the plasma membrane (PM) in eukaryotic cells, but it is unknown how the PM retains sterols at a high concentration....
Sterols are important lipid components of the plasma membrane (PM) in eukaryotic cells, but it is unknown how the PM retains sterols at a high concentration. Phospholipids are asymmetrically distributed in the PM, and phospholipid flippases play an important role in generating this phospholipid asymmetry. Here, we provide evidence that phospholipid flippases are essential for retaining ergosterol in the PM of yeast. A mutant in three flippases, Dnf1-Lem3, Dnf2-Lem3, and Dnf3-Crf1, and a membrane protein, Sfk1, showed a severe growth defect. We recently identified Sfk1 as a PM protein involved in phospholipid asymmetry. The PM of this mutant showed high permeability and low density. Staining with the sterol probe filipin and the expression of a sterol biosensor revealed that ergosterol was not retained in the PM. Instead, ergosterol accumulated in an esterified form in lipid droplets. We propose that ergosterol is retained in the PM by the asymmetrical distribution of phospholipids and the action of Sfk1. Once phospholipid asymmetry is severely disrupted, sterols might be exposed on the cytoplasmic leaflet of the PM and actively transported to the endoplasmic reticulum by sterol transfer proteins.
Topics: ATP-Binding Cassette Transporters; Adenosine Triphosphatases; Cell Membrane; Ergosterol; Membrane Proteins; Membrane Transport Proteins; Phospholipid Transfer Proteins; Repressor Proteins; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins
PubMed: 34038161
DOI: 10.1091/mbc.E20-11-0699 -
Nature Communications Apr 2022In yeast, mitochondria are passed on to daughter cells via the actin cable, motor protein Myo2, and adaptor protein Mmr1. They are released from the actin-myosin...
In yeast, mitochondria are passed on to daughter cells via the actin cable, motor protein Myo2, and adaptor protein Mmr1. They are released from the actin-myosin machinery after reaching the daughter cells. We report that Mmr1 is rapidly degraded by the ubiquitin-proteasome system in Saccharomyces cerevisiae. Redundant ubiquitin ligases Dma1 and Dma2 are responsible for Mmr1 ubiquitination. Dma1/2-mediated Mmr1 ubiquitination requires phosphorylation, most likely at S414 residue by Ste20 and Cla4. These kinases are mostly localized to the growing bud and nearly absent from mother cells, ensuring phosphorylation and ubiquitination of Mmr1 after the mitochondria enter the growing bud. In dma1Δ dma2Δ cells, transported mitochondria are first stacked at the bud-tip and then pulled back to the bud-neck. Stacked mitochondria in dma1Δ dma2Δ cells exhibit abnormal morphology, elevated respiratory activity, and increased level of reactive oxygen species, along with hypersensitivity to oxidative stresses. Collectively, spatiotemporally-regulated Mmr1 turnover guarantees mitochondrial homeostasis.
Topics: Actins; Cell Cycle Proteins; Homeostasis; Mitochondria; Mitochondrial Proteins; Myosins; Proteolysis; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Ubiquitin
PubMed: 35422486
DOI: 10.1038/s41467-022-29704-8 -
Nature Communications Oct 2019Maintenance of cellular proteostasis is achieved by a multi-layered quality control network, which counteracts the accumulation of misfolded proteins by refolding and...
Maintenance of cellular proteostasis is achieved by a multi-layered quality control network, which counteracts the accumulation of misfolded proteins by refolding and degradation pathways. The organized sequestration of misfolded proteins, actively promoted by cellular sequestrases, represents a third strategy of quality control. Here we determine the role of sequestration within the proteostasis network in Saccharomyces cerevisiae and the mechanism by which it occurs. The Hsp42 and Btn2 sequestrases are functionally intertwined with the refolding activity of the Hsp70 system. Sequestration of misfolded proteins by Hsp42 and Btn2 prevents proteostasis collapse and viability loss in cells with limited Hsp70 capacity, likely by shielding Hsp70 from misfolded protein overload. Btn2 has chaperone and sequestrase activity and shares features with small heat shock proteins. During stress recovery Btn2 recruits the Hsp70-Hsp104 disaggregase by directly interacting with the Hsp70 co-chaperone Sis1, thereby shunting sequestered proteins to the refolding pathway.
Topics: Amino Acid Transport Systems; HSP40 Heat-Shock Proteins; HSP70 Heat-Shock Proteins; Heat-Shock Proteins; Protein Refolding; Proteostasis; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins
PubMed: 31649258
DOI: 10.1038/s41467-019-12868-1 -
Life Science Alliance Feb 2021Homologous chromosomes pair with each other during meiosis, culminating in the formation of the synaptonemal complex (SC), which is coupled with meiotic recombination....
Homologous chromosomes pair with each other during meiosis, culminating in the formation of the synaptonemal complex (SC), which is coupled with meiotic recombination. In this study, we showed that a meiosis-specific depletion mutant of a cullin (Cdc53) in the SCF (Skp-Cullin-F-box) ubiquitin ligase, which plays a critical role in cell cycle regulation during mitosis, is deficient in SC formation. However, the mutant is proficient in forming crossovers, indicating the uncoupling of meiotic recombination with SC formation in the mutant. Furthermore, the deletion of the gene encoding a meiosis-specific AAA+ ATPase suppresses SC-assembly defects induced by depletion. On the other hand, the double mutant is defective in meiotic crossover formation, suggesting the assembly of SC with unrepaired DNA double-strand breaks. A temperature-sensitive mutant of , which encodes an F-box protein of SCF, shows meiotic defects similar to those of the -depletion mutant. These results suggest that SCF, probably SCF-dependent protein ubiquitylation, regulates and collaborates with Pch2 in SC assembly and meiotic recombination.
Topics: Cell Cycle Checkpoints; Cell Cycle Proteins; Cullin Proteins; F-Box Proteins; Gene Deletion; Meiosis; Mutation; Recombination, Genetic; Saccharomyces cerevisiae Proteins; Synaptonemal Complex; Ubiquitin-Protein Ligases
PubMed: 33293336
DOI: 10.26508/lsa.202000933 -
FEBS Letters Sep 2017Rab5 GTPases are master regulators of early endosome biogenesis and transport. The genome of Saccharomyces cerevisiae encodes three Rab5 proteins: Vps21, the major...
Rab5 GTPases are master regulators of early endosome biogenesis and transport. The genome of Saccharomyces cerevisiae encodes three Rab5 proteins: Vps21, the major isoform, Ypt52 and Ypt53. Here, we show that Vps21 is the most abundant Rab5 protein and Ypt53 is the least abundant. In stressed cells, Ypt53 levels increase but never exceed that of Vps21. Its induction requires the transcription factors Crz1 and Gis1. In growing cells, the expression of Ypt53 is suppressed by post-transcriptional mechanisms mediated by the untranslated regions of the YPT53 mRNA. Based on genetic experiments, these sequences appear to stimulate deadenylation, Pat1-activated decapping and Xrn1-mediated mRNA degradation. Once this regulation is bypassed, Ypt53 protein levels surpass Vps21, and Ypt53 is sufficient to maintain endosomal function and cell growth.
Topics: Blotting, Western; DNA-Binding Proteins; Endosomes; Histone Demethylases; Microscopy, Fluorescence; RNA, Messenger; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Transcription Factors; rab GTP-Binding Proteins; rab5 GTP-Binding Proteins
PubMed: 28792590
DOI: 10.1002/1873-3468.12785 -
The Journal of Biological Chemistry Apr 2023To cope with an increased external osmolarity, the budding yeast Saccharomyces cerevisiae activates the Hog1 mitogen-activated protein kinase (MAPK) through the...
To cope with an increased external osmolarity, the budding yeast Saccharomyces cerevisiae activates the Hog1 mitogen-activated protein kinase (MAPK) through the high-osmolarity glycerol (HOG) pathway, which governs adaptive responses to osmostress. In the HOG pathway, two apparently redundant upstream branches, termed SLN1 and SHO1, activate cognate MAP3Ks (MAPKK kinase) Ssk2/22 and Ste11, respectively. These MAP3Ks, when activated, phosphorylate and thus activate the Pbs2 MAP2K (MAPK kinase), which in turn phosphorylates and activates Hog1. Previous studies have shown that protein tyrosine phosphatases and the serine/threonine protein phosphatases type 2C negatively regulate the HOG pathway to prevent its excessive and inappropriate activation, which is detrimental to cell growth. The tyrosine phosphatases Ptp2 and Ptp3 dephosphorylate Hog1 at Tyr-176, whereas the protein phosphatase type 2Cs Ptc1 and Ptc2 dephosphorylate Hog1 at Thr-174. In contrast, the identities of phosphatases that dephosphorylate Pbs2 remained less clear. Here, we examined the phosphorylation status of Pbs2 at the activating phosphorylation sites Ser-514 and Thr-518 (S514 and T518) in various mutants, both in the unstimulated and osmostressed conditions. Thus, we found that Ptc1-Ptc4 collectively regulate Pbs2 negatively, but each Ptc acts differently to the two phosphorylation sites in Pbs2. T518 is predominantly dephosphorylated by Ptc1, while S514 can be dephosphorylated by any of Ptc1-4 to an appreciable extent. We also show that Pbs2 dephosphorylation by Ptc1 requires the adaptor protein Nbp2 that recruits Ptc1 to Pbs2, thus highlighting the complex processes involved in regulating adaptive responses to osmostress.
Topics: Glycerol; Intracellular Signaling Peptides and Proteins; MAP Kinase Kinase Kinases; Mitogen-Activated Protein Kinase Kinases; Osmolar Concentration; Phosphoprotein Phosphatases; Phosphorylation; Protein Kinases; Protein Phosphatase 2C; Protein Tyrosine Phosphatases; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Signal Transduction
PubMed: 36870684
DOI: 10.1016/j.jbc.2023.104569 -
Nucleic Acids Research Feb 2016Proteomic and RNomic approaches have identified many components of different ribonucleoprotein particles (RNPs), yet still little is known about the organization and...
Proteomic and RNomic approaches have identified many components of different ribonucleoprotein particles (RNPs), yet still little is known about the organization and protein proximities within these heterogeneous and highly dynamic complexes. Here we describe a targeted cross-linking approach, which combines cross-linking from a known anchor site with affinity purification and mass spectrometry (MS) to identify the changing vicinity interactomes along RNP maturation pathways. Our method confines the reaction radius of a heterobifunctional cross-linker to a specific interaction surface, increasing the probability to capture low abundance conformations and transient vicinal interactors too infrequent for identification by traditional cross-linking-MS approaches, and determine protein proximities within RNPs. Applying the method to two conserved RNA-associated complexes in Saccharomyces cerevisae, the mRNA export receptor Mex67:Mtr2 and the pre-ribosomal Nop7 subcomplex, we identified dynamic vicinal interactomes within those complexes and along their changing pathway milieu. Our results therefore show that this method provides a new tool to study the changing spatial organization of heterogeneous dynamic RNP complexes.
Topics: Cross-Linking Reagents; Electrophoresis, Polyacrylamide Gel; Heterogeneous-Nuclear Ribonucleoproteins; Mass Spectrometry; Membrane Transport Proteins; Models, Molecular; Multiprotein Complexes; Nuclear Proteins; Nucleocytoplasmic Transport Proteins; Protein Binding; Protein Interaction Mapping; Protein Interaction Maps; Protein Structure, Tertiary; Proteome; Proteomics; RNA-Binding Proteins; Reproducibility of Results; Saccharomyces cerevisiae Proteins
PubMed: 26657640
DOI: 10.1093/nar/gkv1366 -
Protein phosphatase 2A (PP2A) promotes anaphase entry after DNA replication stress in budding yeast.Molecular Biology of the Cell Dec 2021DNA replication stress activates the S-phase checkpoint that arrests the cell cycle, but it is poorly understood how cells recover from this arrest. Cyclin-dependent...
DNA replication stress activates the S-phase checkpoint that arrests the cell cycle, but it is poorly understood how cells recover from this arrest. Cyclin-dependent kinase (CDK) and protein phosphatase 2A (PP2A) are key cell cycle regulators, and Cdc55 is a regulatory subunit of PP2A in budding yeast. We found that yeast cells lacking functional PP2A showed slow growth in the presence of hydroxyurea (HU), a DNA synthesis inhibitor, without obvious viability loss. Moreover, PP2A mutants exhibited delayed anaphase entry and sustained levels of anaphase inhibitor Pds1 after HU treatment. A DNA damage checkpoint Chk1 phosphorylates and stabilizes Pds1. We show that and mutation of the Chk1 phosphorylation sites in Pds1 largely restored efficient anaphase entry in PP2A mutants after HU treatment. In addition, deletion of , which encodes the inhibitory kinase for CDK or mutation of the Swe1 phosphorylation site in CDK (), also suppressed the anaphase entry delay in PP2A mutants after HU treatment. Our genetic data suggest that Swe1/CDK acts upstream of Pds1. Surprisingly, showed significant suppression to the viability loss of S-phase checkpoint mutants during DNA synthesis block. Together, our results uncover a PP2A-Swe1-CDK-Chk1-Pds1 axis that promotes recovery from DNA replication stress.
Topics: Anaphase; CDC2 Protein Kinase; Cell Cycle Proteins; Checkpoint Kinase 1; DNA Replication; Green Fluorescent Proteins; Hydroxyurea; Microorganisms, Genetically-Modified; Mutation; Phosphorylation; Protein Phosphatase 2; Protein-Tyrosine Kinases; S Phase; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Securin
PubMed: 34668760
DOI: 10.1091/mbc.E21-04-0222 -
PLoS Genetics Jan 2018Defects in the genes encoding the Paf1 complex can cause increased genome instability. Loss of Paf1, Cdc73, and Ctr9, but not Rtf1 or Leo1, caused increased accumulation...
Defects in the genes encoding the Paf1 complex can cause increased genome instability. Loss of Paf1, Cdc73, and Ctr9, but not Rtf1 or Leo1, caused increased accumulation of gross chromosomal rearrangements (GCRs). Combining the cdc73Δ mutation with individual deletions of 43 other genes, including TEL1 and YKU80, which are involved in telomere maintenance, resulted in synergistic increases in GCR rates. Whole genome sequence analysis of GCRs indicated that there were reduced relative rates of GCRs mediated by de novo telomere additions and increased rates of translocations and inverted duplications in cdc73Δ single and double mutants. Analysis of telomere lengths and telomeric gene silencing in strains containing different combinations of cdc73Δ, tel1Δ and yku80Δ mutations suggested that combinations of these mutations caused increased defects in telomere maintenance. A deletion analysis of Cdc73 revealed that a central 105 amino acid region was necessary and sufficient for suppressing the defects observed in cdc73Δ strains; this region was required for the binding of Cdc73 to the Paf1 complex through Ctr9 and for nuclear localization of Cdc73. Taken together, these data suggest that the increased GCR rate of cdc73Δ single and double mutants is due to partial telomere dysfunction and that Ctr9 and Paf1 play a central role in the Paf1 complex potentially by scaffolding the Paf1 complex subunits or by mediating recruitment of the Paf1 complex to the different processes it functions in.
Topics: Cell Cycle Proteins; DNA-Binding Proteins; Genomic Instability; Intracellular Signaling Peptides and Proteins; Nuclear Proteins; Organisms, Genetically Modified; Phenotype; Protein Binding; Protein Serine-Threonine Kinases; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Telomere; Telomere Homeostasis; Transcriptional Elongation Factors
PubMed: 29320491
DOI: 10.1371/journal.pgen.1007170