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Cells Nov 2019Diverse signals and stress factors regulate the activity and homeostasis of ribosomes in all cells. The protein Asc1/yRACK1 occupies an exposed site at the head region...
Diverse signals and stress factors regulate the activity and homeostasis of ribosomes in all cells. The protein Asc1/yRACK1 occupies an exposed site at the head region of the 40S ribosomal subunit () and represents a central hub for signaling pathways. Asc1 strongly affects protein phosphorylation and is involved in quality control pathways induced by translation elongation arrest. Therefore, it is important to understand the dynamics of protein formations in the Asc1 microenvironment at the . We made use of the in vivo protein-proximity labeling technique Biotin IDentification (BioID). Unbiased proxiOMICs from two adjacent perspectives identified nucleocytoplasmic shuttling mRNA-binding proteins, the deubiquitinase complex Ubp3-Bre5, as well as the ubiquitin E3 ligase Hel2 as neighbors of Asc1. We observed Asc1-dependency of localization of mRNA-binding proteins and the Ubp3 co-factor Bre5. Hel2 and Ubp3-Bre5 are described to balance the mono-ubiquitination of Rps3 (uS3) during ribosome quality control. Here, we show that the absence of Asc1 resulted in massive exposure and accessibility of the C-terminal tail of its ribosomal neighbor Rps3 (uS3). Asc1 and some of its direct neighbors together might form a ribosomal decision tree that is tightly connected to close-by signaling modules.
Topics: Adaptor Proteins, Signal Transducing; Endopeptidases; GTP-Binding Proteins; RNA, Messenger; Receptors for Activated C Kinase; Ribosomal Proteins; Ribosomes; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Signal Transduction; Ubiquitin-Protein Ligases; Ubiquitination
PubMed: 31689955
DOI: 10.3390/cells8111384 -
Current Genetics Apr 2021Under thermal stress, different protein quality control (PQC) strategies are activated to maintain an intact proteome, which may vary from one model system to another.... (Review)
Review
Under thermal stress, different protein quality control (PQC) strategies are activated to maintain an intact proteome, which may vary from one model system to another. Hence thermo-sensitive proteins that lose their active conformation might be refolded with the aid of chaperones or removed by the ubiquitin-proteasome system or the process of autophagy. We have recently developed thermo-sensitive reporters to study PQC in fission yeast and shown the relevance of a third adaptation strategy: the sequestration of misfolded proteins into inclusions which will prevent a rapid degradation and allow the refolding once stress ends. These protein inclusions, protein aggregate centers (PACs), contain a broad spectrum of misfolding/aggregation-prone proteins and chaperones involved in their assembly or dissolution. The chaperone couple Mas5/Ssa2 plays a crucial role in PAC formation, whereas the Hsp104 chaperone promotes their disassembly. The absence of aggregates observed in cells lacking Mas5 could be also explained by the activation of the transcription factor Hsf1 and the induction of chaperone genes, we have excluded this possibility here demonstrating that increased Hsf1 activity and the subsequent overexpression of chaperones do not prevent the assembly of protein aggregates. Protein deposition at certain locations also constitutes a tactic to inactivate proteins temporally. This is the case of Pyp1, the main phosphatase of the stress response kinase Sty1. Upon stress imposition, misfolded Pyp1 is sequestered into cytosolic protein foci while active Sty1 at the nucleus switches on the transcriptional response. In conclusion, we propose that the assembly of aggregation-like foci, PACs in fission yeast, is a crucial PQC strategy during heat stress, and that the Hsp40 chaperone Mas5 is required for PAC assembly and connects physiological and heat-shock triggered PQC.
Topics: HSP40 Heat-Shock Proteins; HSP70 Heat-Shock Proteins; Heat-Shock Proteins; Heat-Shock Response; Molecular Chaperones; Phosphoric Monoester Hydrolases; Protein Folding; Protein Serine-Threonine Kinases; Protein-Tyrosine Kinases; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins
PubMed: 33386485
DOI: 10.1007/s00294-020-01135-2 -
The Journal of Cell Biology May 2022Autophagy is a conserved eukaryotic lysosomal degradation pathway that responds to environmental and cellular cues. Autophagy is essential for the meiotic exit and...
Autophagy is a conserved eukaryotic lysosomal degradation pathway that responds to environmental and cellular cues. Autophagy is essential for the meiotic exit and sporulation in budding yeast, but the underlying molecular mechanisms remain unknown. Here, we show that autophagy is maintained during meiosis and stimulated in anaphase I and II. Cells with higher levels of autophagy complete meiosis faster, and genetically enhanced autophagy increases meiotic kinetics and sporulation efficiency. Strikingly, our data reveal that the conserved phosphatase Cdc14 regulates meiosis-specific autophagy. Cdc14 is activated in anaphase I and II, accompanying its subcellular relocation from the nucleolus to the cytoplasm, where it dephosphorylates Atg13 to stimulate Atg1 kinase activity and thus autophagy. Together, our findings reveal a meiosis-tailored mechanism that spatiotemporally controls meiotic autophagy activity to ensure meiosis progression, exit, and sporulation.
Topics: Adaptor Proteins, Signal Transducing; Anaphase; Autophagy; Autophagy-Related Proteins; Cell Cycle Proteins; Meiosis; Protein Tyrosine Phosphatases; Saccharomyces cerevisiae Proteins
PubMed: 35238874
DOI: 10.1083/jcb.202107151 -
Molecular and Cellular Biology Jun 2020Upstream activation factor (UAF) is a multifunctional transcription factor in that plays dual roles in activating RNA polymerase I (Pol I) transcription and repression...
Upstream activation factor (UAF) is a multifunctional transcription factor in that plays dual roles in activating RNA polymerase I (Pol I) transcription and repression of Pol II. For Pol I, UAF binds to a specific upstream element in the ribosomal DNA (rDNA) promoter and interacts with two other Pol I initiation factors, the TATA-binding protein (TBP) and core factor (CF). We used an integrated combination of chemical cross-linking mass spectrometry (CXMS), molecular genetics, protein biochemistry, and structural modeling to understand the topological framework responsible for UAF complex formation. Here, we report the molecular topology of the UAF complex, describe new structural and functional domains that play roles in UAF complex integrity, assembly, and biological function, and provide roles for previously identified UAF domains that include the Rrn5 SANT and histone fold domains. We highlight the role of new domains in Uaf30 that include an N-terminal winged helix domain and a disordered tethering domain as well as a BORCS6-like domain found in Rrn9. Together, our results reveal a unique network of topological features that coalesce around a histone tetramer-like core to form the dual-function UAF complex.
Topics: Cross-Linking Reagents; DNA-Binding Proteins; Mass Spectrometry; Models, Molecular; Protein Domains; Protein Subunits; RNA Polymerase I; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Transcription Factors; Transcriptional Activation
PubMed: 32253346
DOI: 10.1128/MCB.00056-20 -
RNA (New York, N.Y.) Apr 2021Ribosomal RNA (rRNA) carries extensive 2'-O-methyl marks at functionally important sites. This simple chemical modification is thought to confer stability, promote RNA...
Ribosomal RNA (rRNA) carries extensive 2'-O-methyl marks at functionally important sites. This simple chemical modification is thought to confer stability, promote RNA folding, and contribute to generate a heterogenous ribosome population with a yet-uncharacterized function. 2'-O-methylation occurs both in archaea and eukaryotes and is accomplished by the Box C/D RNP enzyme in an RNA-guided manner. Extensive and partially conflicting structural information exists for the archaeal enzyme, while no structural data is available for the eukaryotic enzyme. The yeast Box C/D RNP consists of a guide RNA, the RNA-primary binding protein Snu13, the two scaffold proteins Nop56 and Nop58, and the enzymatic module Nop1. Here we present the high-resolution structure of the eukaryotic Box C/D methyltransferase Nop1 from bound to the amino-terminal domain of Nop56. We discuss similarities and differences between the interaction modes of the two proteins in archaea and eukaryotes and demonstrate that eukaryotic Nop56 recruits the methyltransferase to the Box C/D RNP through a protein-protein interface that differs substantially from the archaeal orthologs. This study represents a first achievement in understanding the evolution of the structure and function of these proteins from archaea to eukaryotes.
Topics: Amino Acid Sequence; Archaeal Proteins; Binding Sites; Chromosomal Proteins, Non-Histone; Crystallography, X-Ray; Gene Expression; Methylation; Models, Molecular; Nuclear Proteins; Protein Binding; Protein Conformation, alpha-Helical; Protein Conformation, beta-Strand; Protein Interaction Domains and Motifs; Pyrococcus furiosus; RNA, Fungal; RNA, Ribosomal; RNA, Small Nucleolar; Recombinant Proteins; Ribonucleoproteins, Small Nuclear; Ribonucleoproteins, Small Nucleolar; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Sequence Alignment; Structural Homology, Protein; RNA, Guide, CRISPR-Cas Systems
PubMed: 33483369
DOI: 10.1261/rna.077396.120 -
International Review of Cell and... 2020Phosphatidylserine (PS) is an anionic phospholipid that is usually localized in the inner leaflets of the plasma membrane. However, the enzyme scramblase catalyzes the... (Review)
Review
Phosphatidylserine (PS) is an anionic phospholipid that is usually localized in the inner leaflets of the plasma membrane. However, the enzyme scramblase catalyzes the externalization of PS on the outer leaflet of the plasma membrane during apoptosis or cellular stress. This event prompts the recognition of PS displaying cells by phagocytes leading to "apoptotic clearance." Multiple PS receptors (PSRs) mediate this process including members from the TAM (Tyro3, Axl, Mertk) receptor Tyrosine kinases (RTKs) by interacting with PS via bridging proteins like Gas6 and ProS1. Ironically, this network (PS/TAM) that evolved for boosting cellular health through clearance of apoptotic and necrotic cells, has been manoeuvred by pathogens and tumor cells using "apoptotic mimicry." Enveloped viruses, responsible for most of the lethal epidemics and pandemics including the current SARS-CoV2 outbreak, have employed apoptotic mimicry to their advantage. In the current chapter, we summarize the existing knowledge regarding the involvement of PS/Gas6, ProS1/TAM in facilitating infectivity in a diverse set of cell lines, animals as well as organoids. This network executes a largely proviral role in facilitating infection as seen with Zika, Ebola, Influenza and Dengue viruses. However, this response varies with strains and the cells infected, and in some cases, this same signaling displays an antiviral function. We also report multiple studies that have used neutralizing antibodies and small molecule inhibitors in successfully reducing viral replication and ameliorating pathogenicity. Knowledge about this unique signaling pathway and measures that can be taken to inhibit it is most valuable now given how enveloped viruses lead to plagues on the entire globe.
Topics: Animals; Humans; Intercellular Signaling Peptides and Proteins; Protein S; Proto-Oncogene Proteins; RNA Virus Infections; RNA Viruses; Receptor Protein-Tyrosine Kinases; Receptors, Cell Surface; Signal Transduction; c-Mer Tyrosine Kinase; Axl Receptor Tyrosine Kinase
PubMed: 33234246
DOI: 10.1016/bs.ircmb.2020.09.003 -
MSystems Dec 2022A protein's function depends on functional residues that determine its binding specificity or its catalytic activity, but these residues are typically not considered...
A protein's function depends on functional residues that determine its binding specificity or its catalytic activity, but these residues are typically not considered when annotating a protein's function. To help biologists investigate the functional residues of proteins, we developed two interactive web-based tools, SitesBLAST and Sites on a Tree. Given a protein sequence, SitesBLAST finds homologs that have known functional residues and shows whether the functional residues are conserved. Sites on a Tree shows how functional residues vary across a protein family by showing them on a phylogenetic tree. These tools are available at http://papers.genomics.lbl.gov/sites. For most microbes of interest, a genome sequence is available, but the function of its proteins is not known. Instead, proteins' functions are predicted from their similarity to other protein sequences. Within a protein's sequence, a few key residues are most important for function, such as catalyzing a chemical reaction or determining what it binds. But most function prediction tools do not take these key residues into account. We developed interactive tools for identifying functional residues in a protein sequence by comparing it to proteins with known functional residues. Our tools also make it easy to compare key residues across many similar proteins. This should help biologists check if a protein's function is predicted correctly, or to predict if groups of similar proteins have conserved functions.
Topics: Phylogeny; Computational Biology; Proteins; Amino Acid Sequence; Data Interpretation, Statistical
PubMed: 36374048
DOI: 10.1128/msystems.00705-22 -
Biochimica Et Biophysica Acta.... Dec 2022Most eukaryotic secretory and membrane proteins are funneled by the Sec61 complex into the secretory pathway. Furthermore, some substrate peptides rely on two essential...
Most eukaryotic secretory and membrane proteins are funneled by the Sec61 complex into the secretory pathway. Furthermore, some substrate peptides rely on two essential accessory proteins, Sec62 and Sec63, being present to assist with their translocation via the Sec61 channel in post-translational translocation. Cryo-electron microscopy (cryo-EM) recently succeeded in determining atomistic structures of unbound and signal sequence-engaged Sec complexes from Saccharomyces cerevisiae, involving the Sec61 channel and the proteins Sec62, Sec63, Sec71 and Sec72. In this study, we investigated the conformational effects of Sec62 on Sec61. Indeed, we observed in molecular dynamics simulations that the conformational dynamics of lateral gate, plug and pore region of Sec61 are altered by the presence/absence of Sec62. In molecular dynamics simulations that were started from the cryo-EM structures of Sec61 coordinated to Sec62 or of apo Sec61, we observed that the luminal side of the lateral gate gradually adopts a closed conformation similar to the apo state during unbound state simulations. In contrast, it adopts a wider conformation in the bound state. Furthermore, we demonstrate that the conformation of the active (substrate-bound) state of the Sec61 channel shifts toward an alternative conformation in the absence of the substrate. We suggest that the signal peptide holds/stabilizes the active state conformation of Sec61 during post-translational translocation. Thus, our study explains the effect of Sec62 on the conformation of the Sec61 channel and describes the conformational transitions of Sec61 channel.
Topics: Cryoelectron Microscopy; Endoplasmic Reticulum; Heat-Shock Proteins; Membrane Proteins; Membrane Transport Proteins; Protein Sorting Signals; Protein Transport; SEC Translocation Channels; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins
PubMed: 36116515
DOI: 10.1016/j.bbamem.2022.184050 -
PloS One 2023Ste5 is a prototype of scaffold proteins that regulate activation of mitogen-activated protein kinase (MAPK) cascades in all eukaryotes. Ste5 associates with many...
Ste5 is a prototype of scaffold proteins that regulate activation of mitogen-activated protein kinase (MAPK) cascades in all eukaryotes. Ste5 associates with many proteins including Gβγ (Ste4), Ste11 MAPKKK, Ste7 MAPKK, Fus3 and Kss1 MAPKs, Bem1, Cdc24. Here we show that Ste5 also associates with heat shock protein 70 chaperone (Hsp70) Ssa1 and that Ssa1 and its ortholog Ssa2 are together important for Ste5 function and efficient mating responses. The majority of purified overexpressed Ste5 associates with Ssa1. Loss of Ssa1 and Ssa2 has deleterious effects on Ste5 abundance, integrity, and localization particularly when Ste5 is expressed at native levels. The status of Ssa1 and Ssa2 influences Ste5 electrophoresis mobility and formation of high molecular weight species thought to be phosphorylated, ubiquitinylated and aggregated and lower molecular weight fragments. A Ste5 VWA domain mutant with greater propensity to form punctate foci has reduced predicted propensity to bind Ssa1 near the mutation sites and forms more punctate foci when Ssa1 Is overexpressed, supporting a dynamic protein quality control relationship between Ste5 and Ssa1. Loss of Ssa1 and Ssa2 reduces activation of Fus3 and Kss1 MAPKs and FUS1 gene expression and impairs mating shmoo morphogenesis. Surprisingly, ssa1, ssa2, ssa3 and ssa4 single, double and triple mutants can still mate, suggesting compensatory mechanisms exist for folding. Additional analysis suggests Ssa1 is the major Hsp70 chaperone for the mating and invasive growth pathways and reveals several Hsp70-Hsp90 chaperone-network proteins required for mating morphogenesis.
Topics: Adaptor Proteins, Signal Transducing; HSP70 Heat-Shock Proteins; MAP Kinase Kinase Kinases; Mitogen-Activated Protein Kinases; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins
PubMed: 37851593
DOI: 10.1371/journal.pone.0289339 -
DNA Repair Nov 2022Eukaryotic DNA mismatch repair (MMR) initiates through mispair recognition by the MutS homologs Msh2-Msh6 and Msh2-Msh3 and subsequent recruitment of the MutL homologs...
Eukaryotic DNA mismatch repair (MMR) initiates through mispair recognition by the MutS homologs Msh2-Msh6 and Msh2-Msh3 and subsequent recruitment of the MutL homologs Mlh1-Pms1 (human MLH1-PMS2). In bacteria, MutL is recruited by interactions with the connector domain of one MutS subunit and the ATPase and core domains of the other MutS subunit. Analysis of the S. cerevisiae and human homologs have only identified an interaction between the Msh2 connector domain and Mlh1. Here we investigated whether a conserved Msh6 ATPase/core domain-Mlh1 interaction and an Msh2-Msh6 interaction with Pms1 also act in MMR. Mutations in MLH1 affecting interactions with both the Msh2 and Msh6 interfaces caused MMR defects, whereas equivalent pms1 mutations did not cause MMR defects. Mutant Mlh1-Pms1 complexes containing Mlh1 amino acid substitutions were defective for recruitment to mispaired DNA by Msh2-Msh6, did not support MMR in reconstituted Mlh1-Pms1-dependent MMR reactions in vitro, but were proficient in Msh2-Msh6-independent Mlh1-Pms1 endonuclease activity. These results indicate that Mlh1, the common subunit of the Mlh1-Pms1, Mlh1-Mlh2, and Mlh1-Mlh3 complexes, but not Pms1, is recruited by Msh2-Msh6 through interactions with both of its subunits.
Topics: Adenosine Triphosphatases; DNA; DNA Mismatch Repair; DNA-Binding Proteins; Endonucleases; Humans; Mismatch Repair Endonuclease PMS2; MutL Protein Homolog 1; MutL Proteins; MutS Homolog 2 Protein; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins
PubMed: 36122480
DOI: 10.1016/j.dnarep.2022.103405