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Reed B Wickner - One of the best experts on this subject based on the ideXlab platform.
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innate immunity to prions anti prion systems turn a tsunami of prions into a slow drip
Current Genetics, 2021Co-Authors: Reed B Wickner, Herman K. Edskes, Moonil Son, Madaleine NiznikiewiczAbstract:The yeast prions (infectious proteins) [URE3] and [PSI+] are essentially non-functional (or even toxic) amyloid forms of Ure2p and Sup35p, whose normal function is in nitrogen catabolite repression and translation termination, respectively. Yeast has an array of systems working in normal cells that largely block infection with prions, block most prion formation, cure most nascent prions and mitigate the toxic effects of those prions that escape the first three types of systems. Here we review recent progress in defining these anti-prion systems, how they work and how they are regulated. Polymorphisms of the prion domains partially block infection with prions. Ribosome-associated chaperones ensure proper folding of nascent proteins, thus reducing [PSI+] prion formation and curing many [PSI+] variants that do form. Btn2p is a sequestering protein which gathers [URE3] amyloid filaments to one place in the cells so that the prion is often lost by progeny cells. Proteasome impairment produces massive overexpression of Btn2p and paralog Cur1p, resulting in [URE3] curing. Inversely, increased proteasome activity, by derepression of proteasome component gene transcription or by 60S ribosomal subunit gene mutation, prevents prion curing by Btn2p or Cur1p. The nonsense-mediated decay proteins (Upf1,2,3) cure many nascent [PSI+] variants by associating with Sup35p directly. Normal levels of the disaggregating chaperone Hsp104 can also cure many [PSI+] prion variants. By keeping the cellular levels of certain inositol polyphosphates / pyrophosphates low, Siw14p cures certain [PSI+] variants. It is hoped that exploration of the yeast innate immunity to prions will lead to discovery of similar systems in humans.
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innate immunity to yeast prions btn2p and cur1p curing of the ure3 prion is prevented by 60s ribosomal protein deficiency or ubiquitin proteasome system overactivity
Genetics, 2021Co-Authors: Evgeny E. Bezsonov, Herman K. Edskes, Reed B WicknerAbstract:[URE3] is an amyloid-based prion of Ure2p, a negative regulator of poor nitrogen source catabolism in Saccharomyces cerevisiae. Overproduced Btn2p or its paralog Cur1p, in processes requiring Hsp42, cure the [URE3] prion. Btn2p cures by collecting Ure2p amyloid filaments at one place in the cell. We find that rpl4aΔ, rpl21aΔ, rpl21bΔ, rpl11bΔ, and rpl16bΔ (large ribosomal subunit proteins) or ubr2Δ (ubiquitin ligase targeting Rpn4p, an activator of proteasome genes) reduce curing by overproduced Btn2p or Cur1p. Impaired curing in ubr2Δ or rpl21bΔ is restored by an rpn4Δ mutation. No effect of rps14aΔ or rps30bΔ on curing was observed, indicating that 60S subunit deficiency specifically impairs curing. Levels of Hsp42p, Sis1p, or Btn3p are unchanged in rpl4aΔ, rpl21bΔ, or ubr2Δ mutants. Overproduction of Cur1p or Btn2p was enhanced in rpn4Δ and hsp42Δ mutants, lower in ubr2Δ strains, and restored to above wild-type levels in rpn4Δ ubr2Δ strains. As in the wild-type, Ure2N-GFP colocalizes with Btn2-RFP in rpl4aΔ, rpl21bΔ, or ubr2Δ strains, but not in hsp42Δ. Btn2p/Cur1p overproduction cures [URE3] variants with low seed number, but seed number is not increased in rpl4aΔ, rpl21bΔ or ubr2Δ mutants. Knockouts of genes required for the protein sorting function of Btn2p did not affect curing of [URE3], nor did inactivation of the Hsp104 prion-curing activity. Overactivity of the ubiquitin/proteasome system, resulting from 60S subunit deficiency or ubr2Δ, may impair Cur1p and Btn2p curing of [URE3] by degrading Cur1p, Btn2p or another component of these curing systems.
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hermes transposon mutagenesis shows ure3 prion pathology prevented by a ubiquitin targeting protein evidence for carbon nitrogen assimilation cross talk and a second function for Ure2p in saccharomyces cerevisiae
Genetics, 2018Co-Authors: Herman K. Edskes, Maryam Mukhamedova, Bouke K Edskes, Reed B WicknerAbstract:[URE3] is an amyloid-based prion of Ure2p, a regulator of nitrogen catabolism. While most "variants" of the [URE3] prion are toxic, mild variants that only slightly slow growth are more widely studied. The existence of several antiprion systems suggests that some components may be protecting cells from potential detrimental effects of mild [URE3] variants. Our extensive Hermes transposon mutagenesis showed that disruption of YLR352W dramatically slows the growth of [URE3-1] strains. Ylr352wp is an F-box protein, directing selection of substrates for ubiquitination by a "cullin"-containing E3 ligase. For efficient ubiquitylation, cullin-dependent E3 ubiquitin ligases must be NEDDylated, modified by a ubiquitin-related peptide called NEDD8 (Rub1p in yeast). Indeed, we find that disruption of NEDDylation-related genes RUB1, ULA1, UBA3, and UBC12 is also counterselected in our screen. We find that like ylr352wΔ [URE3] strains, ylr352wΔ ure2Δ strains do not grow on nonfermentable carbon sources. Overexpression of Hap4p, a transcription factor stimulating expression of mitochondrial proteins, or mutation of GLN1, encoding glutamine synthetase, allows growth of ylr352w∆ [URE3] strains on glycerol media. Supplying proline as a nitrogen source shuts off the nitrogen catabolite repression (NCR) function of Ure2p, but does not slow growth of ylr352wΔ strains, suggesting a distinct function of Ure2p in carbon catabolism. Also, gln1 mutations impair NCR, but actually relieve the growth defect of ylr352wΔ [URE3] and ylr352wΔ ure2Δ strains, again showing that loss of NCR is not producing the growth defect and suggesting that Ure2p has another function. YLR352W largely protects cells from the deleterious effects of otherwise mild [URE3] variants or of a ure2 mutation (the latter a rarer event), and we name it LUG1 (lets [URE3]/ure2 grow).
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Yeast and Fungal Prions
Cold Spring Harbor perspectives in biology, 2016Co-Authors: Reed B WicknerAbstract:Yeast and fungal prions are infectious proteins, most being self-propagating amyloids of normally soluble proteins. Their effects range from a very mild detriment to lethal, with specific effects dependent on the prion protein and the specific prion variant ("prion strain"). The prion amyloids of Sup35p, Ure2p, and Rnq1p are in-register, parallel, folded β-sheets, an architecture that naturally suggests a mechanism by which a protein can template its conformation, just as DNA or RNA templates its sequence. Prion propagation is critically affected by an array of chaperone systems, most notably the Hsp104/Hsp70/Hsp40 combination, which is responsible for generating new prion seeds from old filaments. The Btn2/Cur1 antiprion system cures most [URE3] prions that develop, and the Ssb antiprion system blocks [PSI+] generation.
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amyloid of the candida albicans Ure2p prion domain is infectious and has an in register parallel β sheet structure
Biochemistry, 2011Co-Authors: Abbi L Engel, Herman K. Edskes, Frank Shewmaker, Fred Dyda, Reed B WicknerAbstract:Ure2p of Candida albicans (Ure2albicans or CaUre2p) can be a prion in Saccharomyces cerevisiae, but Ure2p of Candida glabrata (Ure2glabrata) cannot, even though the Ure2glabrata N-terminal domain is more similar to that of the S. cerevisiae Ure2p (Ure2cerevisiae) than Ure2albicans is. We show that the N-terminal N/Q-rich prion domain of Ure2albicans forms amyloid that is infectious, transmitting [URE3alb] to S. cerevisiae cells expressing only C. albicans Ure2p. Using solid-state nuclear magnetic resonance of selectively labeled C. albicans Ure2p1–90, we show that this infectious amyloid has an in-register parallel β-sheet structure, like that of the S. cerevisiae Ure2p prion domain and other S. cerevisiae prion amyloids. In contrast, the N/Q-rich N-terminal domain of Ure2glabrata does not readily form amyloid, and that formed upon prolonged incubation is not infectious.
Ulrich Baxa - One of the best experts on this subject based on the ideXlab platform.
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characterization of β sheet structure in Ure2p1 89 yeast prion fibrils by solid state nuclear magnetic resonance
Biochemistry, 2007Co-Authors: Ulrich Baxa, Alasdair C Steven, Reed B Wickner, D E Anderson, L N Marekov, W M Yau, Robert TyckoAbstract:Residues 1−89 constitute the Asn- and Gln-rich segment of the Ure2p protein and produce the [URE3] prion of Saccharomyces cerevisiae by forming the core of intracellular Ure2p amyloid. We report the results of solid-state nuclear magnetic resonance (NMR) measurements that probe the molecular structure of amyloid fibrils formed by Ure2p1-89 in vitro. Data include measurements of intermolecular magnetic dipole−dipole couplings in samples that are 13C-labeled at specific sites and two-dimensional 15N−13C and 13C−13C NMR spectra of samples that are uniformly 15N- and 13C-labeled. Intermolecular dipole−dipole couplings indicate that the β-sheets in Ure2p1-89 fibrils have an in-register parallel structure. An in-register parallel β-sheet structure permits polar zipper interactions among side chains of Gln and Asn residues and explains the tolerance of [URE3] to scrambling of the sequence in residues 1−89. Two-dimensional NMR spectra of uniformly labeled Ure2p1-89 fibrils, even when fully hydrated, show NMR line...
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characterization of beta sheet structure in Ure2p1 89 yeast prion fibrils by solid state nuclear magnetic resonance
Biochemistry, 2007Co-Authors: Ulrich Baxa, Alasdair C Steven, Reed B Wickner, D E Anderson, L N Marekov, Waiming Yau, Robert TyckoAbstract:Residues 1−89 constitute the Asn- and Gln-rich segment of the Ure2p protein and produce the [URE3] prion of Saccharomyces cerevisiae by forming the core of intracellular Ure2p amyloid. We report the results of solid-state nuclear magnetic resonance (NMR) measurements that probe the molecular structure of amyloid fibrils formed by Ure2p1-89 in vitro. Data include measurements of intermolecular magnetic dipole−dipole couplings in samples that are 13C-labeled at specific sites and two-dimensional 15N−13C and 13C−13C NMR spectra of samples that are uniformly 15N- and 13C-labeled. Intermolecular dipole−dipole couplings indicate that the β-sheets in Ure2p1-89 fibrils have an in-register parallel structure. An in-register parallel β-sheet structure permits polar zipper interactions among side chains of Gln and Asn residues and explains the tolerance of [URE3] to scrambling of the sequence in residues 1−89. Two-dimensional NMR spectra of uniformly labeled Ure2p1-89 fibrils, even when fully hydrated, show NMR line...
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Structure, function, and amyloidogenesis of fungal prions: filament polymorphism and prion variants.
Advances in Protein Chemistry, 2006Co-Authors: Ulrich Baxa, Todd Cassese, Andrey V Kajava, Alasdair C StevenAbstract:Abstract Infectious proteins (prions) became an important medical issue when they were identified as agents of the transmissible spongiform encephalopathies. More recently, prions have been found in fungi and their investigation has been facilitated by greater experimental tractability. In each case, the normal form of the prion protein may be converted into the infectious form (the prion itself) in an autocatalytic process; conversion may either occur spontaneously or by transmission from an already infected cell. Four fungal prion proteins have been studied in some depth—Ure2p, Sup35p, and Rnq1p of Saccharomyces cerevisiae and HET‐s of Podospora anserina. Each has a “prion domain” that governs infectivity and a “functional domain” that contributes the protein's activity in a wild‐type cell, if it has one. This activity is repressed in prion‐infected cells for loss‐of‐activity prions, [URE3] (the prion of Ure2p) and [PSI] (the prion of Sup35p). For gain‐of‐activity prions, [PIN] (the prion of Rnq1p) and [Het‐s] (the prion of HET‐s), the prion domain is also involved in generating a new activity in infected cells. In prion conversion, prion domains polymerize into an amyloid filament, switching from a “natively unfolded” conformation into an amyloid conformation (stable, protease‐resistant, rich in cross‐β structure). For Ure2p and probably also Sup35p, the functional domain retains its globular fold but is inactivated by a steric mechanism. We review the evidence on which this scenario is based with emphasis on filament structure, summarizing current experimental constraints and appraising proposed models. We conclude that the parallel superpleated β‐structure and a specific β‐helical formulation are valid candidates while other proposals are excluded. In both the Ure2p and Sup35p systems, prion domain amyloid filaments exhibit polymorphic variation. However, once a certain structure is nucleated, it is maintained throughout that filament. Electron microscopy of several Ure2p‐related constructs indicates that the basis for polymorphism lies mainly if not entirely in the prion domain. Filament polymorphism appears to underlie the phenomenon of prion “variants” which differ in the severity of their phenotype, that is, for Ure2p and Sup35p, the stringency with which their activity is switched off. We discuss a possible structural basis for this phenomenon.
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a model for Ure2p prion filaments and other amyloids the parallel superpleated β structure
Proceedings of the National Academy of Sciences of the United States of America, 2004Co-Authors: Andrey V Kajava, Ulrich Baxa, Alasdair C StevenAbstract:In its prion form, Ure2p, a regulator of nitrogen catabolism in Saccharomyces cerevisiae, polymerizes into filaments whereby its C-terminal regulatory domain is inactivated but retains its native fold. The filament has an amyloid fibril backbone formed by the Asn-rich, N-terminal, “prion” domain. The prion domain is also capable of forming fibrils when alone or when fused to other proteins. We have developed a model for the fibril that we call a parallel superpleated β-structure. In this model, the prion domain is divided into nine seven-residue segments, each with a four-residue strand and a three-residue turn, that zig-zag in a planar serpentine arrangement. Serpentines are stacked axially, in register, generating an array of parallel β-sheets, with a small and potentially variable left-hand twist. The interior of the filament is mostly stabilized not by packing of apolar side chains but by H-bond networks generated by the stacking of Asn side chains: charged residues are excluded. The model is consistent with current biophysical, biochemical, and structural data (notably, mass-per-unit-length measurements by scanning transmission electron microscopy that gave one subunit rise per 0.47 nm) and is readily adaptable to other amyloids, for instance the core of Sup35p filaments and glutamine expansions in huntingtin.
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architecture of Ure2p prion filaments the n terminal domains form a central core fiber
Journal of Biological Chemistry, 2003Co-Authors: Ulrich Baxa, Reed B Wickner, Kimberly L Taylor, J S Wall, Martha N Simon, Naiqian Cheng, Alasdair C StevenAbstract:The [URE3] prion is an inactive, self-propagating, filamentous form of the Ure2 protein, a regulator of nitrogen catabolism in yeast. The N-terminal "prion" domain of Ure2p determines its in vivo prion properties and in vitro amyloid-forming ability. Here we determined the overall structures of Ure2p filaments and related polymers of the prion domain fused to other globular proteins. Protease digestion of 25-nm diameter Ure2p filaments trimmed them to 4-nm filaments, which mass spectrometry showed to be composed of prion domain fragments, primarily residues approximately 1-70. Fusion protein filaments with diameters of 14-25 nm were also reduced to 4-nm filaments by proteolysis. The prion domain transforms from the most to the least protease-sensitive part upon filament formation in each case, implying that it undergoes a conformational change. Intact filaments imaged by cryo-electron microscopy or after vanadate staining by scanning transmission electron microscopy (STEM) revealed a central 4-nm core with attached globular appendages. STEM mass per unit length measurements of unstained filaments yielded 1 monomer per 0.45 nm in each case. These observations strongly support a unifying model whereby subunits in Ure2p filaments, as well as in fusion protein filaments, are connected by interactions between their prion domains, which form a 4-nm amyloid filament backbone, surrounded by the corresponding C-terminal moieties.
Alasdair C Steven - One of the best experts on this subject based on the ideXlab platform.
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characterization of β sheet structure in Ure2p1 89 yeast prion fibrils by solid state nuclear magnetic resonance
Biochemistry, 2007Co-Authors: Ulrich Baxa, Alasdair C Steven, Reed B Wickner, D E Anderson, L N Marekov, W M Yau, Robert TyckoAbstract:Residues 1−89 constitute the Asn- and Gln-rich segment of the Ure2p protein and produce the [URE3] prion of Saccharomyces cerevisiae by forming the core of intracellular Ure2p amyloid. We report the results of solid-state nuclear magnetic resonance (NMR) measurements that probe the molecular structure of amyloid fibrils formed by Ure2p1-89 in vitro. Data include measurements of intermolecular magnetic dipole−dipole couplings in samples that are 13C-labeled at specific sites and two-dimensional 15N−13C and 13C−13C NMR spectra of samples that are uniformly 15N- and 13C-labeled. Intermolecular dipole−dipole couplings indicate that the β-sheets in Ure2p1-89 fibrils have an in-register parallel structure. An in-register parallel β-sheet structure permits polar zipper interactions among side chains of Gln and Asn residues and explains the tolerance of [URE3] to scrambling of the sequence in residues 1−89. Two-dimensional NMR spectra of uniformly labeled Ure2p1-89 fibrils, even when fully hydrated, show NMR line...
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characterization of beta sheet structure in Ure2p1 89 yeast prion fibrils by solid state nuclear magnetic resonance
Biochemistry, 2007Co-Authors: Ulrich Baxa, Alasdair C Steven, Reed B Wickner, D E Anderson, L N Marekov, Waiming Yau, Robert TyckoAbstract:Residues 1−89 constitute the Asn- and Gln-rich segment of the Ure2p protein and produce the [URE3] prion of Saccharomyces cerevisiae by forming the core of intracellular Ure2p amyloid. We report the results of solid-state nuclear magnetic resonance (NMR) measurements that probe the molecular structure of amyloid fibrils formed by Ure2p1-89 in vitro. Data include measurements of intermolecular magnetic dipole−dipole couplings in samples that are 13C-labeled at specific sites and two-dimensional 15N−13C and 13C−13C NMR spectra of samples that are uniformly 15N- and 13C-labeled. Intermolecular dipole−dipole couplings indicate that the β-sheets in Ure2p1-89 fibrils have an in-register parallel structure. An in-register parallel β-sheet structure permits polar zipper interactions among side chains of Gln and Asn residues and explains the tolerance of [URE3] to scrambling of the sequence in residues 1−89. Two-dimensional NMR spectra of uniformly labeled Ure2p1-89 fibrils, even when fully hydrated, show NMR line...
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Structure, function, and amyloidogenesis of fungal prions: filament polymorphism and prion variants.
Advances in Protein Chemistry, 2006Co-Authors: Ulrich Baxa, Todd Cassese, Andrey V Kajava, Alasdair C StevenAbstract:Abstract Infectious proteins (prions) became an important medical issue when they were identified as agents of the transmissible spongiform encephalopathies. More recently, prions have been found in fungi and their investigation has been facilitated by greater experimental tractability. In each case, the normal form of the prion protein may be converted into the infectious form (the prion itself) in an autocatalytic process; conversion may either occur spontaneously or by transmission from an already infected cell. Four fungal prion proteins have been studied in some depth—Ure2p, Sup35p, and Rnq1p of Saccharomyces cerevisiae and HET‐s of Podospora anserina. Each has a “prion domain” that governs infectivity and a “functional domain” that contributes the protein's activity in a wild‐type cell, if it has one. This activity is repressed in prion‐infected cells for loss‐of‐activity prions, [URE3] (the prion of Ure2p) and [PSI] (the prion of Sup35p). For gain‐of‐activity prions, [PIN] (the prion of Rnq1p) and [Het‐s] (the prion of HET‐s), the prion domain is also involved in generating a new activity in infected cells. In prion conversion, prion domains polymerize into an amyloid filament, switching from a “natively unfolded” conformation into an amyloid conformation (stable, protease‐resistant, rich in cross‐β structure). For Ure2p and probably also Sup35p, the functional domain retains its globular fold but is inactivated by a steric mechanism. We review the evidence on which this scenario is based with emphasis on filament structure, summarizing current experimental constraints and appraising proposed models. We conclude that the parallel superpleated β‐structure and a specific β‐helical formulation are valid candidates while other proposals are excluded. In both the Ure2p and Sup35p systems, prion domain amyloid filaments exhibit polymorphic variation. However, once a certain structure is nucleated, it is maintained throughout that filament. Electron microscopy of several Ure2p‐related constructs indicates that the basis for polymorphism lies mainly if not entirely in the prion domain. Filament polymorphism appears to underlie the phenomenon of prion “variants” which differ in the severity of their phenotype, that is, for Ure2p and Sup35p, the stringency with which their activity is switched off. We discuss a possible structural basis for this phenomenon.
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the parallel superpleated beta structure as a model for amyloid fibrils of human amylin
Journal of Molecular Biology, 2005Co-Authors: Andrey V Kajava, Ueli Aebi, Alasdair C StevenAbstract:Human amylin is a 37 amino acid residue peptide hormone whose fibrillogenesis has been correlated with type 2 diabetes. These fibrils are rope-like bundles of several 5nm diameter protofilaments. Here, we propose, as a model for the protofilament, a variant of the parallel superpleated beta-structure previously derived for amyloid filaments of the yeast prion Ure2p. In the amylin model, individual polypeptides from residues 9 to 37 have a planar S-shaped fold with three beta-strands. These serpentines are stacked in register, with a 0.47 nm axial rise and a small rotational twist per step, generating an array of three parallel beta-sheets in cross-beta conformation. The interior, the two "bays" sandwiched between adjacent sheets, are occupied by non-polar and by polar/uncharged residues that are predicted to form H-bonded ladders, similar to those found in beta-helical proteins. The N-terminal peptide containing a disulfide bond occupies an extraneous peripheral position in the protofilament. The left-handed twist of the beta-sheets is shown to underlie left-handed coiling of amylin protofilaments in fibrils. The model is consistent with current biophysical, biochemical and genetic data and, in particular, affords a plausible explanation for why rodent amylin does not form fibrils.
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a model for Ure2p prion filaments and other amyloids the parallel superpleated β structure
Proceedings of the National Academy of Sciences of the United States of America, 2004Co-Authors: Andrey V Kajava, Ulrich Baxa, Alasdair C StevenAbstract:In its prion form, Ure2p, a regulator of nitrogen catabolism in Saccharomyces cerevisiae, polymerizes into filaments whereby its C-terminal regulatory domain is inactivated but retains its native fold. The filament has an amyloid fibril backbone formed by the Asn-rich, N-terminal, “prion” domain. The prion domain is also capable of forming fibrils when alone or when fused to other proteins. We have developed a model for the fibril that we call a parallel superpleated β-structure. In this model, the prion domain is divided into nine seven-residue segments, each with a four-residue strand and a three-residue turn, that zig-zag in a planar serpentine arrangement. Serpentines are stacked axially, in register, generating an array of parallel β-sheets, with a small and potentially variable left-hand twist. The interior of the filament is mostly stabilized not by packing of apolar side chains but by H-bond networks generated by the stacking of Asn side chains: charged residues are excluded. The model is consistent with current biophysical, biochemical, and structural data (notably, mass-per-unit-length measurements by scanning transmission electron microscopy that gave one subunit rise per 0.47 nm) and is readily adaptable to other amyloids, for instance the core of Sup35p filaments and glutamine expansions in huntingtin.
Howard Bussey - One of the best experts on this subject based on the ideXlab platform.
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completion of the saccharomyces cerevisiae genome sequence allows identification of ktr5 ktr6 and ktr7 and definition of the nine membered kre2 mnt1 mannosyltransferase gene family in this organism
Yeast, 1997Co-Authors: Marc Lussier, Anne Marie Sdicu, Jane Sheraton, Elaine Winnett, Dahn H Vo, Andreas Dusterhoft, Reginald Storms, Howard BusseyAbstract:The KRE2/MNT1 mannosyltransferase gene family of Saccharomyces cerevisiae currently consists of the KRE2, YUR1, KTR1, KTR2, KTR3 and KTR4 genes. All six encode putative type II membrane proteins with a short cytoplasmic N-terminus, a membrane-spanning region and a highly conserved catalytic lumenal domain. Here we report the identification of the three remaining members of this family in the yeast genome. KTR5 corresponds to an open reading frame (ORF) of the left arm of chromosome XIV, and KTR6 and KTR7 to ORFs on the left arms of chromosomes XVI and IX respectively. The KTR5, KTR6 and KTR7 gene products are highly similar to the Kre2p/Mnt1p family members. Initial functional characterization revealed that some mutant yeast strains containing null copies of these genes displayed cell wall phenotypes. None was K1 killer toxin resistant but ktr6 and ktr7 null mutants were found to be hypersensitive and resistant, respectively, to the drug Calcofluor White. The sequences have been deposited in the GenBank data library under Accession Numbers Z71305; U39205; Z46728.©1997 John Wiley & Sons, Ltd.
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ktr1p is an α 1 2 mannosyltransferase of saccharomyces cerevisiae comparison of the enzymic properties of soluble recombinant ktr1p and kre2p mnt1p produced in pichia pastoris
Biochemical Journal, 1997Co-Authors: Pedro A. Romero, Anne Marie Sdicu, Howard Bussey, Marc Lussier, Annette HerscovicsAbstract:: The yeast genome contains a KRE2/MNT1 family of nine related genes with amino acid similarity to the alpha 1,2-mannosyltransferase Kre2p/Mnt1p, the only member of this family whose enzymic properties have been studied. In this study, the enzymic properties of Ktr1p, another member of this family, were studied and compared to those of Kre2p/Mnt1p. Recombinant soluble forms of Kre2p/Mnt1p and Ktr1p lacking their N-terminal regions were expressed as secreted proteins from the methylotrophic yeast Pichia pastoris. After induction with methanol, the medium contained approx, 40 and 400 mg/l of soluble recombinant Kre2p/Mnt1p and Ktr1p respectively. Both recombinant proteins were shown to exhibit alpha 1,2-mannosyltransferase activity. The enzymes have an absolute requirement for Mn2+ and a similar K(m) for mannose (280-350 mM), methyl-alpha-mannoside (60-90 mM) and GDP-mannose (50-90 microM), but the Vmax was approx. 10 times higher for Kre2p/Mnt1p than for Ktr1p. The enzymes have similar substrate specificities and utilize mannose, methyl-alpha-mannoside, alpha-1,2-mannobiose and methyl-alpha-1,2-mannobiose, as well as Man15-30GlcNAc, derived from mnn2 mutant glycoproteins, as substrates. The enzymes do not utilize alpha-1,6-mannobiose, alpha-1,6-mannotriose, alpha-1,6-mannotetraose, mammalian Man9GlcNAc or yeast Man9-10GlcNAc. These results indicate that Kre2p/ Mnt1p and Ktr1p are capable of participating in both N-glycan and O-glycan biosynthesis.
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functional characterization of the yur1 ktr1 and ktr2 genes as members of the yeast kre2 mnt1 mannosyltransferase gene family
Journal of Biological Chemistry, 1996Co-Authors: Marc Lussier, Anne Marie Sdicu, Anne Camirand, Howard BusseyAbstract:Abstract Eukaryotic glycan structures are progressively elaborated in the secretory pathway. Following the addition of a core N-linked carbohydrate in the endoplasmic reticulum, glycoproteins move to the Golgi complex where the elongation of O-linked sugar chains and processing of complex N-linked oligosaccharide structures take place. In order to better define how such post-translational modifications occur, we have been studying a yeast gene family in which at least one member, KRE2/MNT1, is involved in protein glycosylation. The family currently contains five other members: YUR1, KTR1, KTR2, KTR3 and KTR4 (Mallet, L., Bussereau, F., and Jacquet, M.(1994) Yeast 10, 819-831). All encode putative type II membrane proteins with a short cytoplasmic N terminus, a membrane-spanning region, and a highly conserved catalytic lumenal domain. Kre2p/Mnt1p is a α1,2-mannosyltransferase involved in O- and N-linked glycosylation (Hausler, A., Ballou, L., Ballou, C. E., and Robbins, P. W.(1992) Proc. Natl. Acad. Sci. U. S. A. 89, 6846-6850); however, the role of the other proteins has not yet been established. We have carried out a functional analysis of Ktr1p, Ktr2p, and Yur1p. By in vitro assays, Ktr1p, Ktr2p, and Yur1p have been shown to be mannosyltransferases but, in vivo, do not appear to be involved in O-glycosylation. Examination of the electrophoretic mobility of the N-linked modified protein invertase in null mutant strains indicates that Ktr1p, Ktr2p, and Yur1p are involved in N-linked glycosylation, possibly as redundant enzymes. As found with Kre2p (Hill, K., Boone, C., Goebl, M., Puccia, R., Sdicu, A.-M., and Bussey, H.(1992) Genetics 130, 273-283), Ktr1p, Ktr2p, and Yur1p also seem to be implicated in the glycosylation of cell wall mannoproteins, since yeast cells containing different gene disruptions become K1 killer toxin-resistant. Immunofluorescence microscopy reveals that like Kre2p; Ktr1p, Ktr2p and Yur1p are localized in the Golgi complex.
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localization and targeting of the saccharomyces cerevisiae kre2p mnt1p alpha 1 2 mannosyltransferase to a medial golgi compartment
Journal of Cell Biology, 1995Co-Authors: Marc Lussier, Anne Marie Sdicu, Troy Ketela, Howard BusseyAbstract:The yeast Kre2p/Mnt1p alpha 1,2-mannosyltransferase is a type II membrane protein with a short cytoplasmic amino terminus, a membrane-spanning region, and a large catalytic luminal domain containing one N-glycosylation site. Anti-Kre2p/Mnt1p antibodies identify a 60-kD integral membrane protein that is progressively N-glycosylated in an MNN1-dependent manner. Kre2p/Mnt1p is localized in a Golgi compartment that overlaps with that containing the medial-Golgi mannosyltransferase Mnn1p, and distinct from that including the late Golgi protein Kex1p. To determine which regions of Kre2p/Mnt1p are required for Golgi localization, Kre2p/Mnt1p mutant proteins were assembled by substitution of Kre2p domains with equivalent sequences from the vacuolar proteins DPAP B and Pho8p. Chimeric proteins were tested for correct topology, in vitro and in vivo activity, and were localized intracellularly by indirect immunofluorescence. The results demonstrate that the NH2-terminal cytoplasmic domain is necessary for correct Kre2p Golgi localization whereas, the membrane-spanning and stem domains are dispensable. However, in a test of targeting sufficiency, the presence of the entire Kre2p cytoplasmic tail, plus the transmembrane domain and a 36-amino acid residue luminal stem region was required to localize a Pho8p reporter protein to the yeast Golgi.
Robert Tycko - One of the best experts on this subject based on the ideXlab platform.
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Amyloids of Shuffled Prion Domains That Form Prions Have a Parallel In-Register β-Sheet Structure†
Biochemistry, 2008Co-Authors: Frank Shewmaker, Eric D Ross, Robert Tycko, Reed B WicknerAbstract:The [URE3] and [PSI+] prions of Saccharomyces cerevisiae are self-propagating amyloid forms of Ure2p and Sup35p, respectively. The Q/N-rich N-terminal domains of each protein are necessary and sufficient for the prion properties of these proteins, forming in each case their amyloid cores. Surprisingly, shuffling either prion domain, leaving amino acid content unchanged, does not abrogate the ability of the proteins to become prions. The discovery that the amino acid composition of a polypeptide, not the specific sequence order, determines prion capability seems contrary to the standard folding paradigm that amino acid sequence determines protein fold. The shuffleability of a prion domain further suggests that the β-sheet structure is of the parallel in-register type, and indeed, the normal Ure2 and Sup35 prion domains have such a structure. We demonstrate that two shuffled Ure2 prion domains capable of being prions form parallel in-register β-sheet structures, and our data indicate the same conclusion for...
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characterization of β sheet structure in Ure2p1 89 yeast prion fibrils by solid state nuclear magnetic resonance
Biochemistry, 2007Co-Authors: Ulrich Baxa, Alasdair C Steven, Reed B Wickner, D E Anderson, L N Marekov, W M Yau, Robert TyckoAbstract:Residues 1−89 constitute the Asn- and Gln-rich segment of the Ure2p protein and produce the [URE3] prion of Saccharomyces cerevisiae by forming the core of intracellular Ure2p amyloid. We report the results of solid-state nuclear magnetic resonance (NMR) measurements that probe the molecular structure of amyloid fibrils formed by Ure2p1-89 in vitro. Data include measurements of intermolecular magnetic dipole−dipole couplings in samples that are 13C-labeled at specific sites and two-dimensional 15N−13C and 13C−13C NMR spectra of samples that are uniformly 15N- and 13C-labeled. Intermolecular dipole−dipole couplings indicate that the β-sheets in Ure2p1-89 fibrils have an in-register parallel structure. An in-register parallel β-sheet structure permits polar zipper interactions among side chains of Gln and Asn residues and explains the tolerance of [URE3] to scrambling of the sequence in residues 1−89. Two-dimensional NMR spectra of uniformly labeled Ure2p1-89 fibrils, even when fully hydrated, show NMR line...
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characterization of beta sheet structure in Ure2p1 89 yeast prion fibrils by solid state nuclear magnetic resonance
Biochemistry, 2007Co-Authors: Ulrich Baxa, Alasdair C Steven, Reed B Wickner, D E Anderson, L N Marekov, Waiming Yau, Robert TyckoAbstract:Residues 1−89 constitute the Asn- and Gln-rich segment of the Ure2p protein and produce the [URE3] prion of Saccharomyces cerevisiae by forming the core of intracellular Ure2p amyloid. We report the results of solid-state nuclear magnetic resonance (NMR) measurements that probe the molecular structure of amyloid fibrils formed by Ure2p1-89 in vitro. Data include measurements of intermolecular magnetic dipole−dipole couplings in samples that are 13C-labeled at specific sites and two-dimensional 15N−13C and 13C−13C NMR spectra of samples that are uniformly 15N- and 13C-labeled. Intermolecular dipole−dipole couplings indicate that the β-sheets in Ure2p1-89 fibrils have an in-register parallel structure. An in-register parallel β-sheet structure permits polar zipper interactions among side chains of Gln and Asn residues and explains the tolerance of [URE3] to scrambling of the sequence in residues 1−89. Two-dimensional NMR spectra of uniformly labeled Ure2p1-89 fibrils, even when fully hydrated, show NMR line...
