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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 -
Cell Cycle (Georgetown, Tex.) Dec 2021In fission yeast, MBF-dependent transcription is required for cells to complete S phase. The MBF transcription factor is regulated through a complex feedback mechanism...
In fission yeast, MBF-dependent transcription is required for cells to complete S phase. The MBF transcription factor is regulated through a complex feedback mechanism that involves the co-repressors Yox1 and Nrm1 that are loaded onto MBF at the end of S phase, while positive transactivation is achieved through the constitutive binding of the co-activator Rep2. Here we show that Rep2 is required to fully recruit the chromatin remodelers SWI/SNF and RSC to MBF-regulated promoters. On the contrary, Nrm1 and Yox1, when bound to the MBF complex, block the approximation of these chromatin remodelers to MBF-regulated promoters. We propose that SWI/SNF and RSC are recruited to MBF-regulated genes, and RSC together with SAGA complex are important to regulate the G1-to-S transcriptional wave. Mutants of these remodeler complexes are highly sensitive when cells are exposed to insults that challenge DNA synthesis.
Topics: Cell Cycle Proteins; Chromatin; Homeodomain Proteins; Promoter Regions, Genetic; Repressor Proteins; Schizosaccharomyces; Schizosaccharomyces pombe Proteins; Trans-Activators; Transcription Factors
PubMed: 34843421
DOI: 10.1080/15384101.2021.2008203 -
Molecular Biology of the Cell Apr 2018Cdc48/p97 and the ubiquilin family of UBA-UBL proteins are known for their role in the retrotranslocation of damaged proteins from the endoplasmic reticulum. We...
Cdc48/p97 and the ubiquilin family of UBA-UBL proteins are known for their role in the retrotranslocation of damaged proteins from the endoplasmic reticulum. We demonstrate that Cdc48 and the ubiquilin-like proteins in yeast also play a role in the anterograde trafficking of proteins, in this case the vacuolar protease, Cps1.
Topics: Autophagy; Carboxypeptidases; Cell Cycle Proteins; Endoplasmic Reticulum; Multivesicular Bodies; Nuclear Proteins; Nucleocytoplasmic Transport Proteins; Proteasome Endopeptidase Complex; Protein Transport; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Ubiquitin; Valosin Containing Protein; Vesicular Transport Proteins
PubMed: 29444958
DOI: 10.1091/mbc.E17-11-0652 -
BioEssays : News and Reviews in... Apr 2017The ring-shaped ATPase machine, cohesin, regulates sister chromatid cohesion, transcription, and DNA repair by topologically entrapping DNA. Here, we propose a rigid... (Review)
Review
The ring-shaped ATPase machine, cohesin, regulates sister chromatid cohesion, transcription, and DNA repair by topologically entrapping DNA. Here, we propose a rigid scaffold model to explain how the cohesin regulators Pds5 and Wapl release cohesin from chromosomes. Recent studies have established the Smc3-Scc1 interface as the DNA exit gate of cohesin, revealed a requirement for ATP hydrolysis in ring opening, suggested regulation of the cohesin ATPase activity by DNA and Smc3 acetylation, and provided insights into how Pds5 and Wapl open this exit gate. We hypothesize that Pds5, Wapl, and SA1/2 form a rigid scaffold that docks on Scc1 and anchors the N-terminal domain of Scc1 (Scc1N) to the Smc1 ATPase head. Relative movements between the Smc1-3 ATPase heads driven by ATP and Wapl disrupt the Smc3-Scc1 interface. Pds5 binds the dissociated Scc1N and prolongs this open state of cohesin, releasing DNA. We review the evidence supporting this model and suggest experiments that can further test its key principles.
Topics: Animals; Carrier Proteins; Cell Cycle Proteins; Chromosomal Proteins, Non-Histone; Chromosome Segregation; DNA; DNA Repair; DNA-Binding Proteins; Eukaryota; Humans; Models, Biological; Models, Molecular; Nuclear Proteins; Phosphoproteins; Proto-Oncogene Proteins; Saccharomyces cerevisiae Proteins; Transcription, Genetic; Cohesins
PubMed: 28220956
DOI: 10.1002/bies.201600207 -
PLoS Biology Jul 2019Autophagy recycles cytoplasmic components by sequestering them in double membrane-surrounded autophagosomes. The two proteins Atg11 and Atg17 are scaffolding components...
Autophagy recycles cytoplasmic components by sequestering them in double membrane-surrounded autophagosomes. The two proteins Atg11 and Atg17 are scaffolding components of the Atg1 kinase complex. Atg17 recruits and tethers Atg9-donor vesicles, and the corresponding Atg1 kinase complex induces the formation of nonselective autophagosomes. Atg11 initiates selective autophagy and coordinates the switch to nonselective autophagy by recruiting Atg17. The molecular function of Atg11 remained, however, less well understood. Here, we demonstrate that Atg11 is activated by cargo through a direct interaction with autophagy receptors. Activated Atg11 dimerizes and tethers Atg9 vesicles, which leads to the nucleation of phagophores in direct vicinity of cargo. Starvation reciprocally regulates the activity of both tethering factors by initiating the degradation of Atg11 while Atg17 is activated. This allows Atg17 to sequester and tether Atg9 vesicles independent of cargo to nucleate nonselective phagophores. Our data reveal insights into the molecular mechanisms governing cargo selection and specificity in autophagy.
Topics: Autophagosomes; Autophagy; Autophagy-Related Proteins; Binding, Competitive; Membrane Proteins; Protein Binding; Protein Multimerization; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Unilamellar Liposomes; Vesicular Transport Proteins
PubMed: 31356628
DOI: 10.1371/journal.pbio.3000377 -
Journal of Molecular Biology Mar 2015Mitochondria are the central hub of key cellular processes such as energy conversion, cell signaling, cell cycle regulation and cell differentiation. Therefore, in...
Mitochondria are the central hub of key cellular processes such as energy conversion, cell signaling, cell cycle regulation and cell differentiation. Therefore, in particular, mitochondrial biogenesis and protein translocation have been the focus of intense research for now nearly half a century. In spite of remarkable progress the field has made, many of the proposed mechanisms remain controversial and none of the translocation pathways is yet understood at the high-resolution level. In this context, the present article is intended to identify and discuss current major open questions and unresolved issues in the field in hope that it will stimulate and engage the pursuit of current efforts and expose new directions.
Topics: Animals; Carrier Proteins; Cytosol; Humans; Intracellular Signaling Peptides and Proteins; Membrane Proteins; Mitochondria; Mitochondrial Precursor Protein Import Complex Proteins; Mitochondrial Proteins; Multiprotein Complexes; Protein Precursors; Protein Transport; Saccharomyces cerevisiae Proteins; Tumor Suppressor Proteins
PubMed: 25676309
DOI: 10.1016/j.jmb.2015.02.001 -
Cell Reports Nov 2019Temporal control over protein phosphorylation and dephosphorylation is crucial for accurate chromosome segregation and for completion of the cell division cycle during...
Temporal control over protein phosphorylation and dephosphorylation is crucial for accurate chromosome segregation and for completion of the cell division cycle during exit from mitosis. In budding yeast, the Cdc14 phosphatase is thought to be a major regulator at this time, while in higher eukaryotes PP2A phosphatases take a dominant role. Here, we use time-resolved phosphoproteome analysis in budding yeast to evaluate the respective contributions of Cdc14, PP2A, and PP2A. This reveals an overlapping requirement for all three phosphatases during mitotic progression. Our time-resolved phosphoproteome resource reveals how Cdc14 instructs the sequential pattern of phosphorylation changes, in part through preferential recognition of serine-based cyclin-dependent kinase (Cdk) substrates. PP2A and PP2A in turn exhibit a broad substrate spectrum with some selectivity for phosphothreonines and a role for PP2A in sustaining Aurora kinase activity. These results illustrate synergy and coordination between phosphatases as they orchestrate phosphoproteome dynamics during mitotic progression.
Topics: Cell Cycle Proteins; Mitosis; Phosphoproteins; Protein Phosphatase 2; Protein Tyrosine Phosphatases; Proteome; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins
PubMed: 31722221
DOI: 10.1016/j.celrep.2019.10.041