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Nature Communications May 2020RNase P and MRP are highly conserved, multi-protein/RNA complexes with essential roles in processing ribosomal and tRNAs. Three proteins found in both complexes, Pop1,...
RNase P and MRP are highly conserved, multi-protein/RNA complexes with essential roles in processing ribosomal and tRNAs. Three proteins found in both complexes, Pop1, Pop6, and Pop7 are also telomerase-associated. Here, we determine how temperature sensitive POP1 and POP6 alleles affect yeast telomerase. At permissive temperatures, mutant Pop1/6 have little or no effect on cell growth, global protein levels, the abundance of Est1 and Est2 (telomerase proteins), and the processing of TLC1 (telomerase RNA). However, in pop mutants, TLC1 is more abundant, telomeres are short, and TLC1 accumulates in the cytoplasm. Although Est1/2 binding to TLC1 occurs at normal levels, Est1 (and hence Est3) binding is highly unstable. We propose that Pop-mediated stabilization of Est1 binding to TLC1 is a pre-requisite for formation and nuclear localization of the telomerase holoenzyme. Furthermore, Pop proteins affect TLC1 and the RNA subunits of RNase P/MRP in very different ways.
Topics: Cell Nucleus; DNA-Binding Proteins; Methylation; Protein Binding; RNA; RNA 3' End Processing; Ribonuclease P; Ribonucleoproteins; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Telomerase; Telomere
PubMed: 32358529
DOI: 10.1038/s41467-020-15875-9 -
International Journal of Molecular... Apr 2023Living organisms on the surface biosphere are periodically yet consistently exposed to light. The adaptive or protective evolution caused by this source of energy has... (Review)
Review
Living organisms on the surface biosphere are periodically yet consistently exposed to light. The adaptive or protective evolution caused by this source of energy has led to the biological systems present in a large variety of organisms, including fungi. Among fungi, yeasts have developed essential protective responses against the deleterious effects of light. Stress generated by light exposure is propagated through the synthesis of hydrogen peroxide and mediated by regulatory factors that are also involved in the response to other stressors. These have included Msn2/4, Crz1, Yap1, and Mga2, thus suggesting that light stress is a common factor in the yeast environmental response.
Topics: DNA-Binding Proteins; Saccharomyces cerevisiae Proteins; Transcription Factors; Saccharomyces cerevisiae; Yeasts; Membrane Proteins
PubMed: 37108091
DOI: 10.3390/ijms24086929 -
PLoS Genetics Dec 2020The first metastable assembly intermediate of the eukaryotic ribosomal small subunit (SSU) is the SSU Processome, a large complex of RNA and protein factors that is...
The first metastable assembly intermediate of the eukaryotic ribosomal small subunit (SSU) is the SSU Processome, a large complex of RNA and protein factors that is thought to represent an early checkpoint in the assembly pathway. Transition of the SSU Processome towards continued maturation requires the removal of the U3 snoRNA and biogenesis factors as well as ribosomal RNA processing. While the factors that drive these events are largely known, how they do so is not. The methyltransferase Bud23 has a role during this transition, but its function, beyond the nonessential methylation of ribosomal RNA, is not characterized. Here, we have carried out a comprehensive genetic screen to understand Bud23 function. We identified 67 unique extragenic bud23Δ-suppressing mutations that mapped to genes encoding the SSU Processome factors DHR1, IMP4, UTP2 (NOP14), BMS1 and the SSU protein RPS28A. These factors form a physical interaction network that links the binding site of Bud23 to the U3 snoRNA and many of the amino acid substitutions weaken protein-protein and protein-RNA interactions. Importantly, this network links Bud23 to the essential GTPase Bms1, which acts late in the disassembly pathway, and the RNA helicase Dhr1, which catalyzes U3 snoRNA removal. Moreover, particles isolated from cells lacking Bud23 accumulated late SSU Processome factors and ribosomal RNA processing defects. We propose a model in which Bud23 dissociates factors surrounding its binding site to promote SSU Processome progression.
Topics: DEAD-box RNA Helicases; GTP-Binding Proteins; Methyltransferases; Mutation; Nuclear Proteins; Protein Binding; RNA, Small Nucleolar; RNA-Binding Proteins; Ribosomal Proteins; Ribosome Subunits, Small; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins
PubMed: 33306676
DOI: 10.1371/journal.pgen.1009215 -
Nature Mar 2020The conserved yeast E3 ubiquitin ligase Bre1 and its partner, the E2 ubiquitin-conjugating enzyme Rad6, monoubiquitinate histone H2B across gene bodies during the...
The conserved yeast E3 ubiquitin ligase Bre1 and its partner, the E2 ubiquitin-conjugating enzyme Rad6, monoubiquitinate histone H2B across gene bodies during the transcription cycle. Although processive ubiquitination might-in principle-arise from Bre1 and Rad6 travelling with RNA polymerase II, the mechanism of H2B ubiquitination across genic nucleosomes remains unclear. Here we implicate liquid-liquid phase separation as the underlying mechanism. Biochemical reconstitution shows that Bre1 binds the scaffold protein Lge1, which possesses an intrinsically disordered region that phase-separates via multivalent interactions. The resulting condensates comprise a core of Lge1 encapsulated by an outer catalytic shell of Bre1. This layered liquid recruits Rad6 and the nucleosomal substrate, which accelerates the ubiquitination of H2B. In vivo, the condensate-forming region of Lge1 is required to ubiquitinate H2B in gene bodies beyond the +1 nucleosome. Our data suggest that layered condensates of histone-modifying enzymes generate chromatin-associated 'reaction chambers', with augmented catalytic activity along gene bodies. Equivalent processes may occur in human cells, and cause neurological disease when impaired.
Topics: Biocatalysis; Histones; Intrinsically Disordered Proteins; Microbial Viability; Nucleosomes; Phase Transition; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Transcription Factors; Ubiquitin; Ubiquitin-Conjugating Enzymes; Ubiquitination
PubMed: 32214243
DOI: 10.1038/s41586-020-2097-z -
The Journal of Biological Chemistry Nov 2021Dynamic actin filaments are required for the formation and internalization of endocytic vesicles. Yeast actin cables serve as a track for the translocation of endocytic...
Dynamic actin filaments are required for the formation and internalization of endocytic vesicles. Yeast actin cables serve as a track for the translocation of endocytic vesicles to early endosomes, but the molecular mechanisms regulating the interaction between vesicles and the actin cables remain ambiguous. Previous studies have demonstrated that the yeast Eps15-like protein Pan1p plays an important role in this interaction, and that interaction is not completely lost even after deletion of the Pan1p actin-binding domain, suggesting that additional proteins mediate association of the vesicle with the actin cable. Other candidates for mediating the interaction are endocytic coat proteins Sla2p (yeast Hip1R) and Ent1p/2p (yeast epsins), as these proteins can bind to both the plasma membrane and the actin filament. Here, we investigated the degree of redundancy in the actin-binding activities of Pan1p, Sla2p, and Ent1p/2p involved in the internalization and transport of endocytic vesicles. Expression of the nonphosphorylatable form of Pan1p, Pan1-18TA, caused abnormal accumulation of both actin cables and endocytic vesicles, and this accumulation was additively suppressed by deletion of the actin-binding domains of both Pan1p and Ent1p. Interestingly, deletion of the actin-binding domains of Pan1p and Ent1p in cells lacking the ENT2 gene resulted in severely defective internalization of endocytic vesicles and recruitment of actin cables to the site of endocytosis. These results suggest that Pan1p and Ent1p/2p cooperatively regulate the interaction between the endocytic vesicle and the actin cable.
Topics: Adaptor Proteins, Vesicular Transport; Cell Membrane; Cytoskeletal Proteins; Microfilament Proteins; Protein Domains; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Transport Vesicles; Vesicular Transport Proteins
PubMed: 34592316
DOI: 10.1016/j.jbc.2021.101254 -
Scientific Reports Sep 20166AP and GA are potent inhibitors of yeast and mammalian prions and also specific inhibitors of PFAR, the protein-folding activity borne by domain V of the large rRNA of...
6AP and GA are potent inhibitors of yeast and mammalian prions and also specific inhibitors of PFAR, the protein-folding activity borne by domain V of the large rRNA of the large subunit of the ribosome. We therefore explored the link between PFAR and yeast prion [PSI(+)] using both PFAR-enriched mutants and site-directed methylation. We demonstrate that PFAR is involved in propagation and de novo formation of [PSI(+)]. PFAR and the yeast heat-shock protein Hsp104 partially compensate each other for [PSI(+)] propagation. Our data also provide insight into new functions for the ribosome in basal thermotolerance and heat-shocked protein refolding. PFAR is thus an evolutionarily conserved cell component implicated in the prion life cycle, and we propose that it could be a potential therapeutic target for human protein misfolding diseases.
Topics: Guanabenz; Heat-Shock Proteins; Mutation; Peptide Termination Factors; Phenanthridines; Prions; Protein Folding; RNA, Ribosomal; Ribosomes; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins
PubMed: 27633137
DOI: 10.1038/srep32117 -
Journal of Cell Science Jul 2020PP2A (the form of protein phosphatase 2A containing Cdc55) regulates cell cycle progression by reversing cyclin-dependent kinase (CDK)- and polo-like kinase...
PP2A (the form of protein phosphatase 2A containing Cdc55) regulates cell cycle progression by reversing cyclin-dependent kinase (CDK)- and polo-like kinase (Cdc5)-dependent phosphorylation events. In , Cdk1 phosphorylates securin (Pds1), which facilitates Pds1 binding and inhibits separase (Esp1). During anaphase, Esp1 cleaves the cohesin subunit Scc1 and promotes spindle elongation. Here, we show that PP2A directly dephosphorylates Pds1 both and Pds1 hyperphosphorylation in a deletion mutant enhanced the Pds1-Esp1 interaction, which played a positive role in Pds1 nuclear accumulation and in spindle elongation. We also show that nuclear PP2A plays a role during replication stress to inhibit spindle elongation. This pathway acted independently of the known Mec1, Swe1 or spindle assembly checkpoint (SAC) checkpoint pathways. We propose a model where Pds1 dephosphorylation by PP2A disrupts the Pds1-Esp1 protein interaction and inhibits Pds1 nuclear accumulation, which prevents spindle elongation, a process that is elevated during replication stress.
Topics: Cell Cycle Proteins; Chromosome Segregation; Nuclear Proteins; Phosphorylation; Protein Phosphatase 2; Protein Serine-Threonine Kinases; Protein-Tyrosine Kinases; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Securin; Separase; Spindle Apparatus
PubMed: 32591482
DOI: 10.1242/jcs.243766 -
Nucleic Acids Research Apr 2022Restricting the localization of CENP-A (Cse4 in Saccharomyces cerevisiae) to centromeres prevents chromosomal instability (CIN). Mislocalization of overexpressed CENP-A...
Restricting the localization of CENP-A (Cse4 in Saccharomyces cerevisiae) to centromeres prevents chromosomal instability (CIN). Mislocalization of overexpressed CENP-A to non-centromeric chromatin contributes to CIN in budding and fission yeasts, flies, and humans. Overexpression and mislocalization of CENP-A is observed in cancers and is associated with increased invasiveness. Mechanisms that remove mislocalized CENP-A and target it for degradation have not been defined. Here, we report that Cdc48 and its cofactors Ufd1 and Npl4 facilitate the removal of mislocalized Cse4 from non-centromeric chromatin. Defects in removal of mislocalized Cse4 contribute to lethality of overexpressed Cse4 in cdc48,ufd1 andnpl4 mutants. High levels of polyubiquitinated Cse4 and mislocalization of Cse4 are observed in cdc48-3, ufd1-2 and npl4-1mutants even under normal physiological conditions, thereby defining polyubiquitinated Cse4 as the substrate of the ubiquitin directed segregase Cdc48Ufd1/Npl4. Accordingly, Npl4, the ubiquitin binding receptor, associates with mislocalized Cse4, and this interaction is dependent on Psh1-mediated polyubiquitination of Cse4. In summary, we provide the first evidence for a mechanism that facilitates the removal of polyubiquitinated and mislocalized Cse4 from non-centromeric chromatin. Given the conservation of Cdc48Ufd1/Npl4 in humans, it is likely that defects in such pathways may contribute to CIN in human cancers.
Topics: Centromere; Centromere Protein A; Chromatin; Chromosomal Proteins, Non-Histone; DNA-Binding Proteins; Histones; Humans; Nucleocytoplasmic Transport Proteins; Proteolysis; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Ubiquitin; Valosin Containing Protein; Vesicular Transport Proteins
PubMed: 35234920
DOI: 10.1093/nar/gkac135 -
ELife Sep 2014The TAM receptor tyrosine kinases Tyro3, Axl, and Mer regulate key features of cellular physiology, yet the differential activities of the TAM ligands Gas6 and Protein S...
The TAM receptor tyrosine kinases Tyro3, Axl, and Mer regulate key features of cellular physiology, yet the differential activities of the TAM ligands Gas6 and Protein S are poorly understood. We have used biochemical and genetic analyses to delineate the rules for TAM receptor-ligand engagement and find that the TAMs segregate into two groups based on ligand specificity, regulation by phosphatidylserine, and function. Tyro3 and Mer are activated by both ligands but only Gas6 activates Axl. Optimal TAM signaling requires coincident TAM ligand engagement of both its receptor and the phospholipid phosphatidylserine (PtdSer): Gas6 lacking its PtdSer-binding 'Gla domain' is significantly weakened as a Tyro3/Mer agonist and is inert as an Axl agonist, even though it binds to Axl with wild-type affinity. In two settings of TAM-dependent homeostatic phagocytosis, Mer plays a predominant role while Axl is dispensable, and activation of Mer by Protein S is sufficient to drive phagocytosis.
Topics: Animals; Bone Marrow Cells; Cell Line; Embryo, Mammalian; Female; Fibroblasts; Gene Expression Regulation; HEK293 Cells; Humans; Intercellular Signaling Peptides and Proteins; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Phagocytosis; Phosphatidylserines; Primary Cell Culture; Protein S; Proto-Oncogene Proteins; Receptor Protein-Tyrosine Kinases; Recombinant Proteins; Signal Transduction; c-Mer Tyrosine Kinase; Axl Receptor Tyrosine Kinase
PubMed: 25265470
DOI: 10.7554/eLife.03385 -
Molecular Cell Nov 2017Telomere elongation through telomerase enables chromosome survival during cellular proliferation. The conserved multifunctional shelterin complex associates with...
Telomere elongation through telomerase enables chromosome survival during cellular proliferation. The conserved multifunctional shelterin complex associates with telomeres to coordinate multiple telomere activities, including telomere elongation by telomerase. Similar to the human shelterin, fission yeast shelterin is composed of telomeric sequence-specific double- and single-stranded DNA-binding proteins, Taz1 and Pot1, respectively, bridged by Rap1, Poz1, and Tpz1. Here, we report the crystal structure of the fission yeast Tpz1-Poz1-Rap1 complex that provides the structural basis for shelterin bridge assembly. Biochemical analyses reveal that shelterin bridge assembly is a hierarchical process in which Tpz1 binding to Poz1 elicits structural changes in Poz1, allosterically promoting Rap1 binding to Poz1. Perturbation of the cooperative Tpz1-Poz1-Rap1 assembly through mutation of the "conformational trigger" in Poz1 leads to unregulated telomere lengthening. Furthermore, we find that the human shelterin counterparts TPP1-TIN2-TRF2 also assemble hierarchically, indicating cooperativity as a conserved driving force for shelterin assembly.
Topics: Carrier Proteins; Crystallography, X-Ray; DNA-Binding Proteins; Humans; Protein Structure, Quaternary; Schizosaccharomyces; Schizosaccharomyces pombe Proteins; Shelterin Complex; Telomere-Binding Proteins
PubMed: 29149597
DOI: 10.1016/j.molcel.2017.10.032