-
PLoS Genetics Oct 2014Hsp100 family chaperones of microorganisms and plants cooperate with the Hsp70/Hsp40/NEF system to resolubilize and reactivate stress-denatured proteins. In yeast this...
Hsp100 family chaperones of microorganisms and plants cooperate with the Hsp70/Hsp40/NEF system to resolubilize and reactivate stress-denatured proteins. In yeast this machinery also promotes propagation of prions by fragmenting prion polymers. We previously showed the bacterial Hsp100 machinery cooperates with the yeast Hsp40 Ydj1 to support yeast thermotolerance and with the yeast Hsp40 Sis1 to propagate [PSI+] prions. Here we find these Hsp40s similarly directed specific activities of the yeast Hsp104-based machinery. By assessing the ability of Ydj1-Sis1 hybrid proteins to complement Ydj1 and Sis1 functions we show their C-terminal substrate-binding domains determined distinctions in these and other cellular functions of Ydj1 and Sis1. We find propagation of [URE3] prions was acutely sensitive to alterations in Sis1 activity, while that of [PIN+] prions was less sensitive than [URE3], but more sensitive than [PSI+]. These findings support the ideas that overexpressing Ydj1 cures [URE3] by competing with Sis1 for interaction with the Hsp104-based disaggregation machine, and that different prions rely differently on activity of this machinery, which can explain the various ways they respond to alterations in chaperone function.
Topics: Binding Sites; Endopeptidase Clp; Escherichia coli Proteins; Glutathione Peroxidase; HSP40 Heat-Shock Proteins; HSP70 Heat-Shock Proteins; HSP90 Heat-Shock Proteins; Heat-Shock Proteins; Molecular Chaperones; Mutation; Peptide Termination Factors; Prions; Protein Structure, Tertiary; Saccharomyces cerevisiae Proteins
PubMed: 25329162
DOI: 10.1371/journal.pgen.1004720 -
Molecular Biology of the Cell May 2011Yeast Btn2 facilitates the retrieval of specific proteins from late endosomes (LEs) to the Golgi, a process that may be adversely affected in Batten disease patients. We...
Yeast Btn2 facilitates the retrieval of specific proteins from late endosomes (LEs) to the Golgi, a process that may be adversely affected in Batten disease patients. We isolated the putative yeast orthologue of a human complex I deficiency gene, designated here as BTN3, as encoding a Btn2-interacting protein and negative regulator. First, yeast overexpressing BTN3 phenocopy the deletion of BTN2 and mislocalize certain trans-Golgi proteins, like Kex2 and Yif1, to the LE and vacuole, respectively. In contrast, the deletion of BTN3 results in a tighter pattern of protein localization to the Golgi. Second, BTN3 overexpression alters Btn2 localization from the IPOD compartment, which correlates with a sharp reduction in Btn2-mediated [URE3] prion curing. Third, Btn3 and the Snc1 v-SNARE compete for the same binding domain on Btn2, and this competition controls Btn2 localization and function. The inhibitory effects upon protein retrieval and prion curing suggest that Btn3 sequesters Btn2 away from its substrates, thus down-regulating protein trafficking and aggregation. Therefore Btn3 is a novel negative regulator of intracellular protein sorting, which may be of importance in the onset of complex I deficiency and Batten disease in humans.
Topics: Adaptor Proteins, Vesicular Transport; Amino Acid Transport Systems; Down-Regulation; Endosomal Sorting Complexes Required for Transport; Endosomes; Gene Expression Regulation, Fungal; Heat-Shock Proteins; Neuronal Ceroid-Lipofuscinoses; Prions; Proprotein Convertases; Protein Binding; Protein Interaction Domains and Motifs; Protein Transport; Qb-SNARE Proteins; R-SNARE Proteins; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Two-Hybrid System Techniques; Vesicular Transport Proteins
PubMed: 21441304
DOI: 10.1091/mbc.E10-11-0878 -
Proceedings of the National Academy of... Aug 2021The highly conserved multifunctional polymerase-associated factor 1 (Paf1) complex (PAF1C), composed of five core subunits Paf1, Leo1, Ctr9, Cdc73, and Rtf1,...
The highly conserved multifunctional polymerase-associated factor 1 (Paf1) complex (PAF1C), composed of five core subunits Paf1, Leo1, Ctr9, Cdc73, and Rtf1, participates in all stages of transcription and is required for the Rad6/Bre1-mediated monoubiquitination of histone H2B (H2Bub). However, the molecular mechanisms underlying the contributions of the PAF1C subunits to H2Bub are not fully understood. Here, we report that Ctr9, acting as a hub, interacts with the carboxyl-terminal acidic tail of Rad6, which is required for PAF1C-induced stimulation of H2Bub. Importantly, we found that the Ras-like domain of Cdc73 has the potential to accelerate ubiquitin discharge from Rad6 and thus facilitates H2Bub, a process that might be conserved from yeast to humans. Moreover, we found that Rtf1 HMD stimulates H2Bub, probably through accelerating ubiquitin discharge from Rad6 alone or in cooperation with Cdc73 and Bre1, and that the Paf1/Leo1 heterodimer in PAF1C specifically recognizes the histone H3 tail of nucleosomal substrates, stimulating H2Bub. Collectively, our biochemical results indicate that intact PAF1C is required to efficiently stimulate Rad6/Bre1-mediated H2Bub.
Topics: Cell Cycle Proteins; Cloning, Molecular; Escherichia coli; Gene Expression Regulation, Fungal; Histones; Nuclear Proteins; Nucleosomes; Protein Subunits; RNA-Binding Proteins; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; TATA-Box Binding Protein; Transcriptional Elongation Factors; Ubiquitin-Conjugating Enzymes
PubMed: 34385316
DOI: 10.1073/pnas.2025291118 -
Current Biology : CB Aug 2001The microtubule cytoskeleton plays an important role in cell polarity. Central to this process in fission yeast is tea1p, a marker of polarized cell growth that is... (Review)
Review
The microtubule cytoskeleton plays an important role in cell polarity. Central to this process in fission yeast is tea1p, a marker of polarized cell growth that is delivered to the cell surface in a microtubule-dependent fashion. Recent studies suggest that the actin-binding protein bud6p may be a tea1p effector.
Topics: Cell Polarity; Fungal Proteins; Microfilament Proteins; Microtubule-Associated Proteins; Saccharomyces cerevisiae Proteins; Schizosaccharomyces; Schizosaccharomyces pombe Proteins
PubMed: 11516965
DOI: 10.1016/s0960-9822(01)00362-1 -
Molecular and Cellular Biology Jul 1999In response to nitrogen starvation, diploid cells of the yeast Saccharomyces cerevisiae differentiate to a filamentous growth form known as pseudohyphal differentiation....
In response to nitrogen starvation, diploid cells of the yeast Saccharomyces cerevisiae differentiate to a filamentous growth form known as pseudohyphal differentiation. Filamentous growth is regulated by elements of the pheromone mitogen-activated protein (MAP) kinase cascade and a second signaling cascade involving the receptor Gpr1, the Galpha protein Gpa2, Ras2, and cyclic AMP (cAMP). We show here that the Gpr1-Gpa2-cAMP pathway signals via the cAMP-dependent protein kinase, protein kinase A (PKA), to regulate pseudohyphal differentiation. Activation of PKA by mutation of the regulatory subunit Bcy1 enhances filamentous growth. Mutation and overexpression of the PKA catalytic subunits reveal that the Tpk2 catalytic subunit activates filamentous growth, whereas the Tpk1 and Tpk3 catalytic subunits inhibit filamentous growth. The PKA pathway regulates unipolar budding and agar invasion, whereas the MAP kinase cascade regulates cell elongation and invasion. Epistasis analysis supports a model in which PKA functions downstream of the Gpr1 receptor and the Gpa2 and Ras2 G proteins. Activation of filamentous growth by PKA does not require the transcription factors Ste12 and Tec1 of the MAP kinase cascade, Phd1, or the PKA targets Msn2 and Msn4. PKA signals pseudohyphal growth, in part, by regulating Flo8-dependent expression of the cell surface flocculin Flo11. In summary, the cAMP-dependent protein kinase plays an intimate positive and negative role in regulating filamentous growth, and these findings may provide insight into the roles of PKA in mating, morphogenesis, and virulence in other yeasts and pathogenic fungi.
Topics: Catalytic Domain; Cell Differentiation; Cell Division; Culture Media; Cyclic AMP-Dependent Protein Kinases; DNA-Binding Proteins; Fungal Proteins; GTP-Binding Protein alpha Subunits; GTP-Binding Proteins; Heterotrimeric GTP-Binding Proteins; Membrane Glycoproteins; Membrane Proteins; Nitrogen; Nuclear Proteins; Receptors, Cell Surface; Receptors, G-Protein-Coupled; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Trans-Activators; Transcription Factors; ras Proteins
PubMed: 10373537
DOI: 10.1128/MCB.19.7.4874 -
The Journal of Biological Chemistry Jun 1990To elucidate the mechanism by which C4b-binding protein inhibits the cofactor activity of protein S for anticoagulant-activated protein C, the interactions between...
Inhibition of cofactor activity of protein S by a complex of protein S and C4b-binding protein. Evidence for inactive ternary complex formation between protein S, C4b-binding protein, and activated protein C.
To elucidate the mechanism by which C4b-binding protein inhibits the cofactor activity of protein S for anticoagulant-activated protein C, the interactions between protein S, activated protein C, and C4b-binding protein were studied using solid-phase enzyme immunoassays. Both activated protein C and C4b-binding protein bound to protein S fixed to microplate wells. C4b-binding protein did not inhibit the binding of activated protein C to protein S, nor did activated protein C inhibit the binding of C4b-binding protein to protein S. Activated protein C bound to a protein S-C4b-binding protein complex which was cross-linked with a chemical reagent as well as it bound to free protein S. Protein S-C4b-binding protein complex competitively inhibited activated protein C-binding to free protein S and also the cofactor activity of free protein S. Immunoblotting analysis showed ternary complex formation with protein S, C4b-binding protein, and activated protein C in the liquid phase by treatment with the cross-linking reagent. These findings suggest that the protein S-C4b-binding protein complex inhibits the cofactor activity of free protein S probably by inhibition of functionally active protein S-activated protein C complex formation by the apparent competitive formation of an inactive ternary complex with protein S, C4b-binding protein, and activated protein C.
Topics: Antibodies, Monoclonal; Binding, Competitive; Carrier Proteins; Complement Inactivator Proteins; Cross-Linking Reagents; Electrophoresis, Polyacrylamide Gel; Glycoproteins; Humans; Immunoassay; Immunoblotting; Partial Thromboplastin Time; Protein C; Protein S; Succinimides
PubMed: 2140568
DOI: No ID Found -
The Journal of Biological Chemistry Jan 1994C4b-binding protein (C4BP) down-regulates the anticoagulant cofactor activity of protein S in the protein C pathway since free protein S but not the protein S:C4BP...
C4b-binding protein (C4BP) down-regulates the anticoagulant cofactor activity of protein S in the protein C pathway since free protein S but not the protein S:C4BP complex is anticoagulantly active. To identify beta chain residues responsible for binding protein S, synthetic overlapping pentadecapeptides covering the entire 1-235 sequence were tested as inhibitors of complex formation. The peptide comprising residues 31-45 (VCIKGYHLVGKKTLF) from the first short consensus repeat domain inhibited the binding of C4BP to protein S with half-maximal inhibition at 20-45 microM, and studies suggested the sequence of YxLVG was crucial. Peptide beta(31-45) specifically inhibited the APC cofactor activity of purified protein S in Xa-1-stage coagulation assays with 50% inhibition at 15 microM peptide. Peptide beta(31-45) and related peptides such as beta(34-42) inhibited the binding of protein S to an antipeptide monoclonal antibody made against residues 420-434 of protein S (monoclonal antibody LJ-56). Polyclonal anti-beta(31-45) peptide antibodies inhibited complex formation. Dose-dependent binding studies showed that protein S bound directly to immobilized peptide beta(31-45). These results show that residues 31-45 of the C4BP beta chain provide a binding site for protein S, and they suggest that the C4BP beta chain residues 34-42 are located near residues 420-434 of protein S in the protein S:C4BP complex.
Topics: Amino Acid Sequence; Binding Sites; Blood Coagulation; Carrier Proteins; Complement C4b; Complement Inactivator Proteins; Consensus Sequence; Glycoproteins; Humans; Kinetics; Macromolecular Substances; Molecular Sequence Data; Peptide Fragments; Peptides; Protein S; Structure-Activity Relationship
PubMed: 8300581
DOI: No ID Found -
Blood Oct 1995The complement protein C4b-binding protein plays an important role in the regulation of the protein C anticoagulant pathway. C4b-binding protein can bind to protein S,...
The complement protein C4b-binding protein plays an important role in the regulation of the protein C anticoagulant pathway. C4b-binding protein can bind to protein S, thereby inhibiting the cofactor activity of protein S for activated protein C. In this report, we describe a new role for C4b-binding protein in coagulation. We observed inhibition of the intrinsic factor X activating reaction by the complex of C4b-binding protein and protein S. At the plasma concentration of protein S, the factor X activation was inhibited for 50% and addition of C4b-binding protein led to a potentiation of the inhibition to almost 90%. Because C4b-binding protein alone had no effect on the activation of factor X, we hypothesized that binding of C4b-binding protein to protein S was a prerequisite for optimal inhibition of factor X activation. C4b-binding protein lacking the beta-chain, which is unable to bind to protein S, did not potentiate the inhibitory effect of protein S. In an earlier study, we observed that C4b-binding protein increased the binding affinity of protein S for factor VIII. Therefore, a possible interaction of C4b-binding protein with factor VIII was investigated. C4b-binding protein bound to factor VIII and to thrombin activated factor VIII in a saturable and specific way. Also, factor VIII in complex with von Willebrand factor was able to bind C4b-binding protein. The beta-chain of C4b-binding protein was not required for the interaction with factor VIII because C4b-binding protein lacking the beta-chain also bound to factor VIII. Monoclonal antibodies directed against the alpha-chain of C4b-binding protein inhibited the binding to factor VIII, whereas monoclonal antibodies directed against the beta-chain had no effect on the binding to factor VIII. This finding indicates that the binding site for factor VIII on C4b-binding protein is localized on the alpha-chains of C4b-binding protein. The potentiation by C4b-binding protein of the inhibition of the factor X activation by protein S was blocked by a monoclonal antibody directed against the alpha-chain of C4b-binding protein. This finding indicates that the potentiation of the inhibitory effect of protein S was mediated via an interaction of C4b-binding protein with factor VIII. C4b-binding protein did not bind to factor V and was not able to potentiate the inhibitory effect of protein S on prothrombinase activity.(ABSTRACT TRUNCATED AT 400 WORDS)
Topics: Antibodies, Monoclonal; Binding Sites; Carrier Proteins; Complement Inactivator Proteins; Drug Synergism; Factor IXa; Factor VIII; Factor X; Factor Xa; Glycoproteins; Humans; Kinetics; Protein S; von Willebrand Factor
PubMed: 7670108
DOI: No ID Found -
Nucleic Acids Research Apr 2000Basic helix-loop-helix (bHLH) proteins are among the most well studied and functionally important regulatory proteins in all eukaryotes. The HLH domain dictates... (Review)
Review
Basic helix-loop-helix (bHLH) proteins are among the most well studied and functionally important regulatory proteins in all eukaryotes. The HLH domain dictates dimerization to create homo- and heterodimers. Dimerization juxtaposes the basic regions of the two monomers to create a DNA interaction surface that recognizes the consensus sequence called the E-box, 5'-CANNTG-3'. Several bHLH proteins have been identified in the yeast Saccharomyces cerevisiae using traditional genetic methodologies. These proteins regulate diverse biological pathways. The completed sequence of the yeast genome, combined with novel methodologies allowing whole-genome expression studies, now offers a unique opportunity to study the function of these bHLH proteins. It is the purpose of this review to summarize the current knowledge of bHLH protein function in yeast.
Topics: Amino Acid Sequence; Basic Helix-Loop-Helix Leucine Zipper Transcription Factors; Basic Helix-Loop-Helix Transcription Factors; DNA-Binding Proteins; Fungal Proteins; Genes, Fungal; Helix-Loop-Helix Motifs; Repressor Proteins; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Sequence Homology, Amino Acid; Trans-Activators; Transcription Factors
PubMed: 10710415
DOI: 10.1093/nar/28.7.1499 -
Molecular Cell Sep 2009In this issue of Molecular Cell, Schmidt et al. (2009) untangle the interplay between the COP9 signalosome (CSN), cullin-associated and neddylation-dissociated 1 (CAND1)...
In this issue of Molecular Cell, Schmidt et al. (2009) untangle the interplay between the COP9 signalosome (CSN), cullin-associated and neddylation-dissociated 1 (CAND1) protein, and cullin-RING ubiquitin ligases (CRLs).
Topics: CDC2 Protein Kinase; COP9 Signalosome Complex; Conserved Sequence; Cullin Proteins; Endopeptidases; F-Box Proteins; Metalloproteases; Models, Molecular; Multiprotein Complexes; Peptide Hydrolases; Proline; Protein Binding; Protein Stability; SKP Cullin F-Box Protein Ligases; Schizosaccharomyces; Schizosaccharomyces pombe Proteins; Ubiquitins
PubMed: 19748350
DOI: 10.1016/j.molcel.2009.08.011