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FEBS Letters Oct 2018Mitochondria are organelles containing two membranes that are distinct in composition and function. A role of the mitochondrial outer membrane (MOM) is to mediate...
Mitochondria are organelles containing two membranes that are distinct in composition and function. A role of the mitochondrial outer membrane (MOM) is to mediate contact of the organelle with the rest of the cell. In yeast, the MOM contains about 40 different integral proteins. Recently, we described the MOM protein Mcp3, which can serve as a multicopy suppressor of loss of ERMES complex that mediates mitochondria-endoplasmic reticulum contacts. To shed further light on the role of Mcp3 in the MOM, we analyzed its physical interaction with other proteins. We show that Mcp3 interacts with the MOM protein Om45 and the inner membrane protein Aim19. Our observations hint toward a potential involvement of Mcp3 in a structural and/or functional link between both mitochondrial membranes.
Topics: Endoplasmic Reticulum; Membrane Proteins; Mitochondria; Mitochondrial Membranes; Mitochondrial Precursor Protein Import Complex Proteins; Mitochondrial Proteins; Mutation; Protein Binding; Protein Interaction Maps; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins
PubMed: 30192984
DOI: 10.1002/1873-3468.13243 -
Prion May 2017Yeast prions are protein-based genetic elements that propagate through cell populations via cytosolic transfer from mother to daughter cell. Molecular chaperone proteins...
Yeast prions are protein-based genetic elements that propagate through cell populations via cytosolic transfer from mother to daughter cell. Molecular chaperone proteins including Hsp70, the Hsp40/J-protein Sis1, and Hsp104 are required for continued prion propagation, however the specific requirements of chaperone proteins differ for various prions. We recently reported that Swa2, the yeast homolog of the mammalian protein auxilin, is specifically required for the propagation of the prion [URE3]. [URE3] propagation requires both a functional J-domain and the tetratricopeptide repeat (TPR) domain of Swa2, but does not require Swa2 clathrin binding. We concluded that the TPR domain determines the specificity of the genetic interaction between Swa2 and [URE3], and that this domain likely interacts with one or more proteins with a C-terminal EEVD motif. Here we extend that analysis to incorporate additional data that supports this hypothesis. We also present new data eliminating Hsp104 as the relevant Swa2 binding partner and discuss our findings in the context of other recent work involving Hsp90. Based on these findings, we propose a new model for Swa2's involvement in [URE3] propagation in which Swa2 and Hsp90 mediate the formation of a multi-protein complex that increases the number of sites available for Hsp104 disaggregation.
Topics: Auxilins; Glutathione Peroxidase; HSP40 Heat-Shock Proteins; HSP90 Heat-Shock Proteins; Heat-Shock Proteins; Phosphoproteins; Prions; Protein Binding; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Vesicular Transport Proteins
PubMed: 28574745
DOI: 10.1080/19336896.2017.1331810 -
Nature Communications Aug 2019Stress granules are membraneless protein- and mRNA-rich organelles that form in response to perturbations in environmental conditions. Stress granule formation is...
Stress granules are membraneless protein- and mRNA-rich organelles that form in response to perturbations in environmental conditions. Stress granule formation is reversible, and persistent stress granules have been implicated in a variety of neurodegenerative disorders, including amyotrophic lateral sclerosis. However, characterization of the factors involved in dissolving stress granules is incomplete. Many stress granule proteins contain prion-like domains (PrLDs), some of which have been linked to stress granule formation. Here, we demonstrate that the PrLD-containing yeast protein kinase Sky1 is a stress granule component. Sky1 is recruited to stress granules in part via its PrLD, and Sky1's kinase activity regulates timely stress granule disassembly during stress recovery. This effect is mediated by phosphorylation of the stress granule component Npl3. Sky1 can compensate for defects in chaperone-mediated stress granule disassembly and vice-versa, demonstrating that cells have multiple overlapping mechanisms for re-solubilizing stress granule components.
Topics: Gene Expression Regulation, Fungal; Heat-Shock Proteins; Neurodegenerative Diseases; Nuclear Proteins; Organelles; Phosphorylation; Poly(A)-Binding Proteins; Prions; Protein Domains; Protein Serine-Threonine Kinases; RNA-Binding Proteins; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins
PubMed: 31399582
DOI: 10.1038/s41467-019-11550-w -
Journal of Thrombosis and Haemostasis :... Apr 2019Essentials Protein S and FV-Short are synergistic cofactors to Tissue Factor Pathway Inhibitor α (TFPIα). An assay for the TFPIα synergistic cofactor activity of...
Essentials Protein S and FV-Short are synergistic cofactors to Tissue Factor Pathway Inhibitor α (TFPIα). An assay for the TFPIα synergistic cofactor activity of protein S with FV-Short was developed. The assay was specific for the synergistic TFPIα-cofactor activity of free protein S. Protein S deficient individuals with known mutations were correctly distinguished from controls. SUMMARY: Background Protein S is an anticoagulant cofactor to both activated protein C and tissue factor pathway inhibitor (TFPIα). The TFPIα-cofactor activity of protein S is stimulated by a short isoform of factor V (FV-Short), the two proteins functioning in synergy. Objective Using the synergistic TFPIα-cofactor activity between protein S and FV-Short to develop a functional test for plasma protein S. Patients/Methods TFPIα-mediated inhibition of FXa in the presence of FV-Short, protein S and negatively charged phospholipid vesicles was monitored in time by synthetic substrate S2765. TFPIα, FXa and FV-Short were purified proteins, whereas diluted plasma from protein S deficient patients or controls were used as source for protein S. Results The assay was specific for free protein S demonstrating good correlation to free protein S plasma levels (r = 0.92) with a Y-axis intercept of -5%. Correlation to concentrations of total protein S (free and C4BPβ+-bound) was lower (r = 0.88) and the Y-axis intercept was +46%, which is consistent with the specificity for free protein S. The test distinguished protein S-deficient individuals from 6 families with known ProS1 mutations from family members having no mutation. Protein S levels of warfarin-treated protein S deficient cases were lower than protein S in cases treated with warfarin for other causes. Conclusions We describe a new assay measuring the TFPIα-cofactor activity of plasma protein S. The test identifies type I/III protein S deficiencies and will be a useful tool to detect type II protein S deficiency having defective TFPIα-cofactor activity.
Topics: Adult; Anticoagulants; Calcium-Binding Proteins; Case-Control Studies; Factor V; Female; Humans; Lipoproteins; Male; Middle Aged; Mutation; Peptide Fragments; Predictive Value of Tests; Protein S; Protein S Deficiency; Spectrum Analysis; Warfarin; Young Adult
PubMed: 30740865
DOI: 10.1111/jth.14405 -
PLoS Genetics Jul 2021During meiosis, defects in critical events trigger checkpoint activation and restrict cell cycle progression. The budding yeast Pch2 AAA+ ATPase orchestrates the...
During meiosis, defects in critical events trigger checkpoint activation and restrict cell cycle progression. The budding yeast Pch2 AAA+ ATPase orchestrates the checkpoint response launched by synapsis deficiency; deletion of PCH2 or mutation of the ATPase catalytic sites suppress the meiotic block of the zip1Δ mutant lacking the central region of the synaptonemal complex. Pch2 action enables adequate levels of phosphorylation of the Hop1 axial component at threonine 318, which in turn promotes activation of the Mek1 effector kinase and the ensuing checkpoint response. In zip1Δ chromosomes, Pch2 is exclusively associated to the rDNA region, but this nucleolar fraction is not required for checkpoint activation, implying that another yet uncharacterized Pch2 population must be responsible for this function. Here, we have artificially redirected Pch2 to different subcellular compartments by adding ectopic Nuclear Export (NES) or Nuclear Localization (NLS) sequences, or by trapping Pch2 in an immobile extranuclear domain, and we have evaluated the effect on Hop1 chromosomal distribution and checkpoint activity. We have also deciphered the spatial and functional impact of Pch2 regulators including Orc1, Dot1 and Nup2. We conclude that the cytoplasmic pool of Pch2 is sufficient to support the meiotic recombination checkpoint involving the subsequent Hop1-Mek1 activation on chromosomes, whereas the nuclear accumulation of Pch2 has pathological consequences. We propose that cytoplasmic Pch2 provokes a conformational change in Hop1 that poises it for its chromosomal incorporation and phosphorylation. Our discoveries shed light into the intricate regulatory network controlling the accurate balance of Pch2 distribution among different cellular compartments, which is essential for proper meiotic outcomes.
Topics: Cell Cycle Checkpoints; Cell Membrane; Chromosome Pairing; Chromosomes, Fungal; Cytoplasm; DNA-Binding Proteins; Histone-Lysine N-Methyltransferase; Meiosis; Microorganisms, Genetically-Modified; Nuclear Pore Complex Proteins; Nuclear Proteins; Origin Recognition Complex; Recombination, Genetic; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins
PubMed: 34260586
DOI: 10.1371/journal.pgen.1009560 -
Polyubiquitin and ubiquitin-like signals share common recognition sites on proteasomal subunit Rpn1.The Journal of Biological Chemistry 2021Proteasome-mediated substrate degradation is an essential process that relies on the coordinated actions of ubiquitin (Ub), shuttle proteins containing Ub-like (UBL)...
Proteasome-mediated substrate degradation is an essential process that relies on the coordinated actions of ubiquitin (Ub), shuttle proteins containing Ub-like (UBL) domains, and the proteasome. Proteinaceous substrates are tagged with polyUb and shuttle proteins, and these signals are then recognized by the proteasome, which subsequently degrades the substrate. To date, three proteasomal receptors have been identified, as well as multiple shuttle proteins and numerous types of polyUb chains that signal for degradation. While the components of this pathway are well-known, our understanding of their interplay is unclear-especially in the context of Rpn1, the largest proteasomal subunit. Here, using nuclear magnetic resonance (NMR) spectroscopy in combination with competition assays, we show that Rpn1 associates with UBL-containing proteins and polyUb chains, while exhibiting a preference for shuttle protein Rad23. Rpn1 appears to contain multiple Ub/UBL-binding sites, theoretically as many as one for each of its hallmark proteasome/cyclosome repeats. Remarkably, we also find that binding sites on Rpn1 can be shared among Ub and UBL species, while proteasomal receptors Rpn1 and Rpn10 can compete with each other for binding of shuttle protein Dsk2. Taken together, our results rule out the possibility of exclusive recognition sites on Rpn1 for individual Ub/UBL signals and further emphasize the complexity of the redundancy-laden proteasomal degradation pathway.
Topics: Binding Sites; Cell Cycle Proteins; Cytoplasm; DNA-Binding Proteins; Humans; Magnetic Resonance Spectroscopy; Membrane Proteins; Polyubiquitin; Proteasome Endopeptidase Complex; Protein Binding; Proteolysis; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Ubiquitin; Ubiquitins
PubMed: 33617881
DOI: 10.1016/j.jbc.2021.100450 -
ELife Jun 2019Tail-anchored (TA) proteins insert post-translationally into the endoplasmic reticulum (ER), the outer mitochondrial membrane (OMM) and peroxisomes. Whereas the GET...
Tail-anchored (TA) proteins insert post-translationally into the endoplasmic reticulum (ER), the outer mitochondrial membrane (OMM) and peroxisomes. Whereas the GET pathway controls ER-targeting, no dedicated factors are known for OMM insertion, posing the question of how accuracy is achieved. The mitochondrial AAA-ATPase Msp1 removes mislocalized TA proteins from the OMM, but it is unclear, how Msp1 clients are targeted for degradation. Here we screened for factors involved in degradation of TA proteins mislocalized to mitochondria. We show that the ER-associated degradation (ERAD) E3 ubiquitin ligase Doa10 controls cytoplasmic level of Msp1 clients. Furthermore, we identified the uncharacterized OMM protein Fmp32 and the ectopically expressed subunit of the ER-mitochondria encounter structure (ERMES) complex Gem1 as native clients for Msp1 and Doa10. We propose that productive localization of TA proteins to the OMM is ensured by complex assembly, while orphan subunits are extracted by Msp1 and eventually degraded by Doa10.
Topics: Adenosine Triphosphatases; Anion Transport Proteins; Endoplasmic Reticulum; Membrane Proteins; Mitochondrial Membranes; Mitochondrial Proteins; Monocarboxylic Acid Transporters; Protein Transport; Saccharomyces cerevisiae Proteins; Ubiquitin-Protein Ligases
PubMed: 31172943
DOI: 10.7554/eLife.45506 -
Life Science Alliance Aug 2019Centromeric chromatin in fission yeast is distinguished by the presence of nucleosomes containing the histone H3 variant Cnp1 Cell cycle-specific deposition of Cnp1...
Centromeric chromatin in fission yeast is distinguished by the presence of nucleosomes containing the histone H3 variant Cnp1 Cell cycle-specific deposition of Cnp1 requires the Mis16-Mis18-Mis19 complex, which is thought to direct recruitment of Scm3-chaperoned Cnp1/histone H4 dimers to DNA. Here, we present the structure of the essential Mis18 partner protein Mis19 and describe its interaction with Mis16, revealing a bipartite-binding site. We provide data on the stoichiometry and overall architecture of the complex and provide detailed insights into the Mis18-Mis19 interface.
Topics: Binding Sites; Carrier Proteins; Centromere; Chromosomal Proteins, Non-Histone; Histones; Models, Molecular; Multiprotein Complexes; Mutation; Protein Binding; Schizosaccharomyces; Schizosaccharomyces pombe Proteins
PubMed: 31371524
DOI: 10.26508/lsa.201900408 -
Cell Jul 2014Polarization of the plasma membrane (PM) into domains is an important mechanism to compartmentalize cellular activities and to establish cell polarity. Polarization...
Polarization of the plasma membrane (PM) into domains is an important mechanism to compartmentalize cellular activities and to establish cell polarity. Polarization requires formation of diffusion barriers that prevent mixing of proteins between domains. Recent studies have uncovered that the endoplasmic reticulum (ER) of budding yeast and neurons is polarized by diffusion barriers, which in neurons controls glutamate signaling in dendritic spines. The molecular identity of these barriers is currently unknown. Here, we show that a direct interaction between the ER protein Scs2 and the septin Shs1 creates the ER diffusion barrier in yeast. Barrier formation requires Epo1, a novel ER-associated subunit of the polarisome that interacts with Scs2 and Shs1. ER-septin tethering polarizes the ER into separate mother and bud domains, one function of which is to position the spindle in the mother until M phase by confining the spindle capture protein Num1 to the mother ER.
Topics: Carrier Proteins; Cell Cycle Proteins; Cell Polarity; Cytoskeletal Proteins; Diffusion; Endoplasmic Reticulum; Membrane Proteins; Nuclear Envelope; S Phase; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins
PubMed: 25083872
DOI: 10.1016/j.cell.2014.06.033 -
Nature Communications Nov 2019Hyperaccumulators typically refer to plants that absorb and tolerate elevated amounts of heavy metals. Due to their unique metal trafficking abilities, hyperaccumulators...
Hyperaccumulators typically refer to plants that absorb and tolerate elevated amounts of heavy metals. Due to their unique metal trafficking abilities, hyperaccumulators are promising candidates for bioremediation applications. However, compared to bacteria-based bioremediation systems, plant life cycle is long and growing conditions are difficult to maintain hindering their adoption. Herein, we combine the robust growth and engineerability of bacteria with the unique waste management mechanisms of plants by using a more tractable platform-the common baker's yeast-to create plant-like hyperaccumulators. Through overexpression of metal transporters and engineering metal trafficking pathways, engineered yeast strains are able to sequester metals at concentrations 10-100 times more than established hyperaccumulator thresholds for chromium, arsenic, and cadmium. Strains are further engineered to be selective for either cadmium or strontium removal, specifically for radioactive Sr. Overall, this work presents a systematic approach for transforming yeast into metal hyperaccumulators that are as effective as their plant counterparts.
Topics: Antiporters; Arsenic; Biodegradation, Environmental; Cadmium; Carrier Proteins; Cation Transport Proteins; Chromium; Copper Transport Proteins; Copper Transporter 1; Iron-Binding Proteins; Membrane Transport Proteins; Metabolic Engineering; Metals, Heavy; SLC31 Proteins; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Strontium; Strontium Radioisotopes
PubMed: 31704944
DOI: 10.1038/s41467-019-13093-6