The Experts below are selected from a list of 360 Experts worldwide ranked by ideXlab platform
Ronald Melki - One of the best experts on this subject based on the ideXlab platform.
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The Yarrowia lipolytica orthologs of Sup35p assemble into thioflavin T-negative amyloid fibrils
Biochemical and Biophysical Research Communications, 2020Co-Authors: Mehdi Kabani, Ronald MelkiAbstract:The translation terminator Sup35p assembles into self-replicating fibrillar aggregates that are responsible for the [PSI þ ] prion state. The Q/N-rich N-terminal domain together with the highly charged middle-domain (NM domain) drive the assembly of Sup35p into amyloid fibrils in vitro. NM domains are highly divergent among yeasts. The ability to convert to a prion form is however conserved among Sup35 orthologs. In particular, the Yarrowia lipolytica Sup35p stands out with an exceptionally high prion conversion rate. In the present work, we show that different Yarrowia lipolytica strains contain one of two Sup35p orthologs that differ by the number of repeats within their NM domain. The Y. lipolytica Sup35 proteins are able to assemble into amyloid fibrils. Contrary to S. cerevisiae Sup35p, fibrils made of full-length or NM domains of Y. lipolytica Sup35 proteins did not bind Thioflavin-T, a well-known marker of amyloid aggregates.
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the amphipathic gm1 molecule stabilizes amyloid aggregates preventing their cytotoxicity
Biophysical Journal, 2020Co-Authors: Monica Bucciantini, Ronald Melki, Manuela Leri, Massimo Stefani, Sandra Zecchiorlandini, Daniele NosiAbstract:Amyloid aggregates have been demonstrated to exert cytotoxic effects in several diseases. It is widely accepted that the complex and fascinating aggregation pathway involves a series of steps during which many heterogeneous intermediates are generated. This process may be greatly potentiated by the presence of amphipathic components of plasma membrane because they may serve as interaction, condensation, and nucleation points. However, there are few data regarding structural alterations induced by the binding between the amyloid fibrils and membrane components and its direct effects on cell integrity. In this study, we found, by 1-anilinonaphthalene 8-sulfonic acid and transmission electron microscopy/fast Fourier transform, that yeast prion Sup35 oligomers showed higher structural uniformity and altered surface properties when grown in the presence of monosialotetrahexosylganglioside, a component of the cell membrane. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and confocal/sensitized Forster resonance energy transfer analyses revealed that these fibrils showed low cytotoxicity and affinity to plasma membrane. Moreover, time-lapse analysis of Sup35 oligomer fibrillation on cells suggested that the amyloid aggregation process per se exerts cytotoxic effects through the interaction of amyloid intermediates with plasma membrane components. These data provide, to our knowledge, new insights to understand the mechanism of amyloid growth and cytotoxicity in the pathogenesis of amyloid diseases.
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Glucose availability dictates the export of the soluble and prion forms of Sup35p via periplasmic or extracellular vesicles
Molecular Microbiology, 2020Co-Authors: Mehdi Kabani, Marion Pilard, Ronald MelkiAbstract:The yeast [PSI$^+$] prion originates from the self‐perpetuating transmissible aggregates of the translation termination factor Sup35p. We previously showed that infectious Sup35p particles are exported outside the cells via extracellular vesicles (EV). This finding suggested a function for EV in the vertical and horizontal transmission of yeast prions. Here we report a significant export of Sup35p within periplasmic vesicles (PV) upon glucose starvation. We show that PV are up to three orders of magnitude more abundant than EV. However, PV and EV are different in terms of size and protein content, and their export is oppositely regulated by glucose availability in the growth medium. Overall, our work suggests that the export of prion particles to both the periplasm and the extracellular space needs to be considered to address the physiological consequences of vesicle‐mediated yeast prions trafficking.
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growth phase dependent changes in the size and infectivity of sds resistant Sup35p assemblies associated with the psi prion in yeast
Molecular Microbiology, 2019Co-Authors: Kai Wang, Ronald Melki, Mehdi KabaniAbstract:The translation termination factor Sup35p can form self-replicating fibrillar aggregates responsible for the [PSI+ ] prion state. Sup35p aggregation yields detergent-resistant assemblies detectable on agarose gels under semi-denaturant conditions and fluorescent puncta within the yeast cytosol when the protein is fused to GFP. It is still unclear whether any of these manifestations of [PSI+ ] truly correspond to the Sup35p assemblies that faithfully transmit the [PSI+ ] prion from mother to daughter cells. The infectious titer of prions in cells can be indirectly assessed by the ability of [PSI+ ] cells lysates to induce the prion state when introduced into naive cells. Here, we report that the dramatic changes in the size and amounts of SDS-resistant Sup35p that occur during growth do not correlate with the infectious titer. Our results suggest that fluorescent Sup35-GFP puncta and detergent-resistant Sup35p assemblies are good indicators of Sup35p conversion to the prion state but not of infectious particles number.
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More than just trash bins? Potential roles for extracellular vesicles in the vertical and horizontal transmission of yeast prions
Current Genetics, 2016Co-Authors: Mehdi Kabani, Ronald MelkiAbstract:In the yeast Saccharomyces cerevisiae , an ensemble of structurally and functionally diverse cytoplasmic proteins has the ability to form self-perpetuating protein aggregates (e.g. prions) which are the vectors of heritable non-Mendelian phenotypic traits. Whether harboring these prions is deleterious—akin to mammalian degenerative disorders—or beneficial—as epigenetic modifiers of gene expression—for yeasts has been intensely debated and strong arguments were made in support of both views. We recently reported that the yeast prion protein Sup35p is exported via extracellular vesicles (EV), both in its soluble and aggregated infectious states. Herein, we discuss the possible implications of this observation and propose several hypotheses regarding the roles of EV in both vertical and horizontal propagation of ‘good’ and ‘bad’ yeast prions.
Reed B Wickner - One of the best experts on this subject based on the ideXlab platform.
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locating folds of the in register parallel β sheet of the Sup35p prion domain infectious amyloid
Proceedings of the National Academy of Sciences of the United States of America, 2014Co-Authors: Anton Gorkovskiy, Robert Tycko, Kent R Thurber, Reed B WicknerAbstract:The [PSI+] prion is a self-propagating amyloid of the translation termination factor, Sup35p, of Saccharomyces cerevisiae. The N-terminal 253 residues (NM) of this 685-residue protein normally function in regulating mRNA turnover but spontaneously form infectious amyloid in vitro. We converted the three Ile residues in Sup35NM to Leu and then replaced 16 single residues with Ile, one by one, and prepared Ile-1-(13)C amyloid of each mutant, seeding with amyloid formed by the reference sequence Sup35NM. Using solid-state NMR, we showed that 10 of the residues examined, including six between residues 30 and 90, showed the ∼0.5-nm distance between labels diagnostic of the in-register parallel amyloid architecture. The five scattered N domain residues with wider spacing may be in turns or loops; one is a control at the C terminus of M. All mutants, except Q56I, showed little or no [PSI+] transmission barrier from the reference sequence, suggesting that they could assume a similar amyloid architecture in vitro when seeded with filaments of reference sequence Sup35NM. Infection of yeast cells expressing the reference SUP35 gene sequence with amyloid of several mutants produced [PSI+] transfectants with similar efficiency as did reference sequence Sup35NM amyloid. Our work provides a stringent demonstration that the Sup35 prion domain has the folded in-register parallel β-sheet architecture and suggests common locations of the folds. This architecture naturally suggests a mechanism of inheritance of conformation, the central mystery of prions.
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sporadic distribution of prion forming ability of Sup35p from yeasts and fungi
Genetics, 2014Co-Authors: Herman K Edskes, Hima J Khamar, Chialin Winchester, Alexandria J Greenler, Albert Zhou, Ryan P Mcglinchey, Anton Gorkovskiy, Reed B WicknerAbstract:Sup35p of Saccharomyces cerevisiae can form the [PSI+] prion, an infectious amyloid in which the protein is largely inactive. The part of Sup35p that forms the amyloid is the region normally involved in control of mRNA turnover. The formation of [PSI+] by Sup35p’s from other yeasts has been interpreted to imply that the prion-forming ability of Sup35p is conserved in evolution, and thus of survival/fitness/evolutionary value to these organisms. We surveyed a larger number of yeast and fungal species by the same criteria as used previously and find that the Sup35p from many species cannot form prions. [PSI+] could be formed by the Sup35p from Candida albicans, Candida maltosa, Debaromyces hansenii, and Kluyveromyces lactis, but orders of magnitude less often than the S. cerevisiae Sup35p converts to the prion form. The Sup35s from Schizosaccharomyces pombe and Ashbya gossypii clearly do not form [PSI+]. We were also unable to detect [PSI+] formation by the Sup35ps from Aspergillus nidulans, Aspergillus fumigatus, Magnaporthe grisea, Ustilago maydis, or Cryptococcus neoformans. Each of two C. albicans SUP35 alleles can form [PSI+], but transmission from one to the other is partially blocked. These results suggest that the prion-forming ability of Sup35p is not a conserved trait, but is an occasional deleterious side effect of a protein domain conserved for another function.
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two prion variants of Sup35p have in register parallel β sheet structures independent of hydration
Biochemistry, 2009Co-Authors: Frank Shewmaker, Robert Tycko, Dmitry Kryndushkin, Bo Chen, Reed B WicknerAbstract:The [PSI+] prion is a self-propagating amyloid of the Sup35 protein, normally a subunit of the translation termination factor, but impaired in this vital function when in the amyloid form. The Sup35 N, M, and C domains are the amino-terminal prion domain, a connecting polar domain, and the essential C-terminal domain resembling eukaryotic elongation factor 1α respectively. Different [PSI+] isolates (prion variants) may have distinct biological properties, associated with different amyloid structures. Here we use solid state NMR to examine the structure of infectious Sup35NM amyloid fibrils of two prion variants. We find that both variants have an in-register parallel β-sheet structure, both in the fully hydrated form and in the lyophilized form. Moreover, we confirm that some leucine residues in the M domain participate in the in-register parallel β-sheet structure. Transmission of the [PSI+] prion by amyloid fibrils of Sup35NM and transmission of the [URE3] prion by amyloid fibrils of recombinant full-le...
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a prion of yeast metacaspase homolog mca1p detected by a genetic screen
Proceedings of the National Academy of Sciences of the United States of America, 2009Co-Authors: Julie Nemecek, Toru Nakayashiki, Reed B WicknerAbstract:Saccharomyces cerevisiae can be infected with four amyloid-based prions: [URE3], [PSI+], [PIN+], and [SWI+], due to self-propagating aggregation of Ure2p, Sup35p, Rnq1p and Swi1p, respectively. We searched for new prions of yeast by fusing random segments of yeast DNA to SUP35MC, encoding the Sup35 protein lacking its own prion domain, selecting clones in which Sup35MC function was impaired. Three different clones contained parts of the Q/N-rich amino-terminal domain of Mca1p/Yca1p with the Sup35 part of the fusion protein partially inactive. This inactivity was dominant, segregated 4:0 in meiosis, and was efficiently transferred by cytoplasmic mixing. The inactivity was cured by overexpression of Hsp104, but the prion could arise again in the cured strain (reversible curing). Overproduction of the Mca1 N-terminal domain induced the de novo appearance of the prion form of the fusion. The prion state, which we name [MCA], was transmitted to the chromosomally encoded Mca1p based on genetic, cytological and biochemical tests.
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amyloid of rnq1p the basis of the pin prion has a parallel in register β sheet structure
Proceedings of the National Academy of Sciences of the United States of America, 2008Co-Authors: Reed B Wickner, Fred Dyda, Robert TyckoAbstract:The [PIN+] prion, a self-propagating amyloid form of Rnq1p, increases the frequency with which the [PSI+] or [URE3] prions arise de novo. Like the prion domains of Sup35p and Ure2p, Rnq1p is rich in N and Q residues, but rnq1Δ strains have no known phenotype except for inability to propagate the [PIN+] prion. We used solid-state NMR methods to examine amyloid formed in vitro from recombinant Rnq1 prion domain (residues 153–405) labeled with Tyr-1–13C (14 residues), Leu-1–13C (7 residues), or Ala-3–13C (13 residues). The carbonyl chemical shifts indicate that most Tyr and Leu residues are in β-sheet conformation. Experiments designed to measure the distance from each labeled residue to the next nearest labeled carbonyl showed that almost all Tyr and Leu carbonyl carbon atoms were ≈0.5 nm from the next nearest Tyr and Leu residues, respectively. This result indicates that the Rnq1 prion domain forms amyloid consisting of parallel β-strands that are either in register or are at most one amino acid out of register. Similar experiments with Ala-3–13C indicate that the β-strands are indeed in-register. The parallel in-register structure, now demonstrated for each of the yeast prions, explains the faithful templating of prion strains, and suggests as well a mechanism for the rare hetero-priming that is [PIN+]'s defining characteristic.
Mehdi Kabani - One of the best experts on this subject based on the ideXlab platform.
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The Yarrowia lipolytica orthologs of Sup35p assemble into thioflavin T-negative amyloid fibrils
Biochemical and Biophysical Research Communications, 2020Co-Authors: Mehdi Kabani, Ronald MelkiAbstract:The translation terminator Sup35p assembles into self-replicating fibrillar aggregates that are responsible for the [PSI þ ] prion state. The Q/N-rich N-terminal domain together with the highly charged middle-domain (NM domain) drive the assembly of Sup35p into amyloid fibrils in vitro. NM domains are highly divergent among yeasts. The ability to convert to a prion form is however conserved among Sup35 orthologs. In particular, the Yarrowia lipolytica Sup35p stands out with an exceptionally high prion conversion rate. In the present work, we show that different Yarrowia lipolytica strains contain one of two Sup35p orthologs that differ by the number of repeats within their NM domain. The Y. lipolytica Sup35 proteins are able to assemble into amyloid fibrils. Contrary to S. cerevisiae Sup35p, fibrils made of full-length or NM domains of Y. lipolytica Sup35 proteins did not bind Thioflavin-T, a well-known marker of amyloid aggregates.
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Glucose availability dictates the export of the soluble and prion forms of Sup35p via periplasmic or extracellular vesicles
Molecular Microbiology, 2020Co-Authors: Mehdi Kabani, Marion Pilard, Ronald MelkiAbstract:The yeast [PSI$^+$] prion originates from the self‐perpetuating transmissible aggregates of the translation termination factor Sup35p. We previously showed that infectious Sup35p particles are exported outside the cells via extracellular vesicles (EV). This finding suggested a function for EV in the vertical and horizontal transmission of yeast prions. Here we report a significant export of Sup35p within periplasmic vesicles (PV) upon glucose starvation. We show that PV are up to three orders of magnitude more abundant than EV. However, PV and EV are different in terms of size and protein content, and their export is oppositely regulated by glucose availability in the growth medium. Overall, our work suggests that the export of prion particles to both the periplasm and the extracellular space needs to be considered to address the physiological consequences of vesicle‐mediated yeast prions trafficking.
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growth phase dependent changes in the size and infectivity of sds resistant Sup35p assemblies associated with the psi prion in yeast
Molecular Microbiology, 2019Co-Authors: Kai Wang, Ronald Melki, Mehdi KabaniAbstract:The translation termination factor Sup35p can form self-replicating fibrillar aggregates responsible for the [PSI+ ] prion state. Sup35p aggregation yields detergent-resistant assemblies detectable on agarose gels under semi-denaturant conditions and fluorescent puncta within the yeast cytosol when the protein is fused to GFP. It is still unclear whether any of these manifestations of [PSI+ ] truly correspond to the Sup35p assemblies that faithfully transmit the [PSI+ ] prion from mother to daughter cells. The infectious titer of prions in cells can be indirectly assessed by the ability of [PSI+ ] cells lysates to induce the prion state when introduced into naive cells. Here, we report that the dramatic changes in the size and amounts of SDS-resistant Sup35p that occur during growth do not correlate with the infectious titer. Our results suggest that fluorescent Sup35-GFP puncta and detergent-resistant Sup35p assemblies are good indicators of Sup35p conversion to the prion state but not of infectious particles number.
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More than just trash bins? Potential roles for extracellular vesicles in the vertical and horizontal transmission of yeast prions
Current Genetics, 2016Co-Authors: Mehdi Kabani, Ronald MelkiAbstract:In the yeast Saccharomyces cerevisiae , an ensemble of structurally and functionally diverse cytoplasmic proteins has the ability to form self-perpetuating protein aggregates (e.g. prions) which are the vectors of heritable non-Mendelian phenotypic traits. Whether harboring these prions is deleterious—akin to mammalian degenerative disorders—or beneficial—as epigenetic modifiers of gene expression—for yeasts has been intensely debated and strong arguments were made in support of both views. We recently reported that the yeast prion protein Sup35p is exported via extracellular vesicles (EV), both in its soluble and aggregated infectious states. Herein, we discuss the possible implications of this observation and propose several hypotheses regarding the roles of EV in both vertical and horizontal propagation of ‘good’ and ‘bad’ yeast prions.
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Sup35p in Its Soluble and Prion States Is Packaged inside Extracellular Vesicles
mBio, 2015Co-Authors: Mehdi Kabani, Ronald MelkiAbstract:ABSTRACT The yeast Saccharomyces cerevisiae harbors several prions that constitute powerful models to investigate the mechanisms of epigenetic structural inheritance. [ PSI + ] is undoubtedly the best-known yeast prion and results from the conversion of the translation termination factor Sup35p into self-perpetuating protein aggregates. Structurally different conformers of Sup35p aggregates can lead to [ PSI + ] strains with weak or strong prion phenotypes. Yeast prions are faithfully transmitted from mother to daughter cells during cell division, upon cytoplasmic mixing during mating, or when Sup35p fibrils made in test tubes are introduced into spheroplasts. Virtually all living cells in the three domains of life, Bacteria , Archaea , and Eukarya , secrete small membrane vesicles in the extracellular space. These extracellular vesicles (EV) have gained increasing interest as vehicles for the intercellular transfer of signaling molecules, nucleic acids, and pathogenic factors, as well as prion-like protein aggregates associated with neurodegenerative diseases. To begin to explore the question of whether EV could represent a natural mean for yeast prion transmission from cell to cell, we purified these extracellular vesicles and assessed whether they contained Sup35p. Here, we show that Sup35p is secreted within EV released in the extracellular medium of yeast cultures. We demonstrate that Sup35p within EV isolated from strong and weak [ PSI + ] cells is in an infectious prion conformation. Among the possible implications of our work is the possibility of previously unsuspected EV-mediated horizontal cell-to-cell transfer of fungal prions. IMPORTANCE Most living cells in the three domains of life, Bacteria , Archaea , and Eukarya , secrete small membrane vesicles in the extracellular space. These extracellular vesicles (EV) were long viewed as “trash cans” by which cells disposed of unwanted macromolecules. EV gained renewed interest as their roles as vehicles for the cell-to-cell transfer of nucleic acids, signaling molecules, and pathogenic factors were recently uncovered. Of particular interest is their proposed role in the prion-like propagation of toxic protein aggregates in neurodegenerative diseases. Yeasts naturally harbor prion proteins that are excellent models to investigate the mechanisms of formation, propagation, and elimination of self-perpetuating protein aggregates. Here we show for the first time that a yeast prion is secreted within EV in its infectious aggregated state. A major implication of our work is the possibility of EV-mediated horizontal spread of fungal prions.
Mick F Tuite - One of the best experts on this subject based on the ideXlab platform.
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The copper transport-associated protein Ctr4 can form prion-like epigenetic determinants in Schizosaccharomyces pombe.
Microbial cell (Graz Austria), 2017Co-Authors: Theodora C. Sideri, Mick F Tuite, Yoko Yashiroda, David A. Ellis, María Rodríguez-lópez, Minoru Yoshida, Jürg BählerAbstract:Prions are protein-based infectious entities associated with fatal brain diseases in animals, but also modify a range of host-cell phenotypes in the budding yeast, Saccharomyces cerevisiae. Many questions remain about the evolution and biology of prions. Although several functionally distinct prion-forming proteins exist in S. cerevisiae, [HET-s] of Podospora anserina is the only other known fungal prion. Here we investigated prion-like, protein-based epigenetic transmission in the fission yeast Schizosaccharomyces pombe. We show that S. pombe cells can support the formation and maintenance of the prion form of the S. cerevisiae Sup35 translation factor [PSI+], and that the formation and propagation of these Sup35 aggregates is inhibited by guanidine hydrochloride, indicating commonalities in prion propagation machineries in these evolutionary diverged yeasts. A proteome-wide screen identified the Ctr4 copper transporter subunit as a putative prion with a predicted prion-like domain. Overexpression of the ctr4 gene resulted in large Ctr4 protein aggregates that were both detergent and proteinase-K resistant. Cells carrying such [CTR+] aggregates showed increased sensitivity to oxidative stress, and this phenotype could be transmitted to aggregate-free [ctr–] cells by transformation with [CTR+] cell extracts. Moreover, this [CTR+] phenotype was inherited in a non-Mendelian manner following mating with naive [ctr–] cells, but intriguingly the [CTR+] phenotype was not eliminated by guanidine-hydrochloride treatment. Thus, Ctr4 exhibits multiple features diagnostic of other fungal prions and is the first example of a prion in fission yeast. These findings suggest that transmissible protein-based determinants of traits may be more widespread among fungi.
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structural definition is important for the propagation of the yeast psi prion
Molecular Cell, 2013Co-Authors: Ricardo Marchante, Michelle L Rowe, Jo Zenthon, Mark J Howard, Mick F TuiteAbstract:Prions are propagated in Saccharomyces cerevisiae with remarkable efficiency, yet we know little about the structural basis of sequence variations in the prion protein that support or prohibit propagation of the prion conformation. We show that certain single-amino-acid substitutions in the prion protein Sup35 impact negatively on the maintenance of the associated prion-based [PSI+] trait by combining in vivo phenotypic analysis with solution NMR structural studies. A clear correlation is observed between mutationally induced conformational differences in one of the oligopeptide repeats (R2) in the N terminus of Sup35 and the relative ability to propagate [PSI+]. Strikingly, substitution of one of a Gly-Gly pair with highly charged residues that significantly increase structural definition of R2 lead to a severe [PSI+] propagation defect. These findings offer a molecular explanation for the dominant-negative effects of such psi-no-more (PNM) mutations and demonstrate directly the importance of localized structural definition in prion propagation.
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The Candida albicans Sup35p protein (CaSup35p): function, prion-like behaviour and an associated polyglutamine length polymorphism
Microbiology, 2002Co-Authors: Catarina G. Resende, Steven N. Parham, Caroline L. Tinsley, Paulo C. Ferreira, Julio A. B. Duarte, Mick F TuiteAbstract:The Sup35p protein of Saccharomyces cerevisiae is an essential translation factor whose prion-like properties give rise to the non-Mendelian genetic element [PSI(+)]. In this study the SUP35 gene from the related yeast species Candida albicans has been characterized. The CaSUP35 gene encodes a protein (CaSup35p) of 729 aa which shows 65% amino acid identity to the S. cerevisiae Sup35p protein (ScSup35p), with the C-terminal region showing greater identity (79%) than the N-terminal region. The full-length CaSup35p can functionally replace ScSup35p in S. cerevisiae although complementation is only complete when CaSup35p is overexpressed. Complementation only requires expression of the CaSup35p C domain. In S. cerevisiae the full-length CaSup35p is unable to establish a prion-like aggregated state even in the presence of endogenous ScSup35p prion 'seeds', thus confirming the existence of a species barrier in fungal prion propagation. Subcellular localization studies in C. albicans show that although CaSup35p is normally ribosome-associated, when not ribosome-associated, it does not form pelletable high-molecular-mass aggregates characteristic of the ScSup35p in [PSI(+)] strains. Unlike the ScSup35p, the CaSup35p N domain contains a number of polyglutamine repeats although it does contain seven copies of the peptide GGYQQ that is repeated in the ScSup35p N domain. Analysis of the CaSUP35 gene from 14 different strains of C. albicans identified four naturally occurring polymorphisms associated with changes in the length of the largest of the polyglutamine repeats. These findings have important implications for the evolution of fungal prion genes.
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oligopeptide repeats in the yeast protein Sup35p stabilize intermolecular prion interactions
The EMBO Journal, 2001Co-Authors: Steven N. Parham, Catarina G. Resende, Mick F TuiteAbstract:The nuclear-encoded Sup35p protein is responsible for the prion-like [PSI(+)] determinant of yeast, with Sup35p existing largely as a high molecular weight aggregate in [PSI(+)] strains. Here we show that the five oligopeptide repeats present at the N-terminus of Sup35p are responsible for stabilizing aggregation of Sup35p in vivo. Sequential deletion of the oligopeptide repeats prevented the maintenance of [PSI(+)] by the truncated Sup35p, although deletants containing only two repeats could be incorporated into pre-existing aggregates of wild-type Sup35p. The mammalian prion protein PrP also contains similar oligopeptide repeats and we show here that a human PrP repeat (PHGGGWGQ) is able functionally to replace a Sup35p oligopeptide repeat to allow stable [PSI(+)] propagation in vivo. Our data suggest a model in which the oligopeptide repeats in Sup35p stabilize intermolecular interactions between Sup35p proteins that initiate establishment of the aggregated state. Modulating repeat number therefore alters the rate of yeast prion conversion in vivo. Furthermore, there appears to be evolutionary conservation of function of the N-terminally located oligopeptide repeats in prion propagation.
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a conditional lethal translation termination defect in a sup45 mutant of the yeast succhuromyces cerevisiue
FEBS Journal, 1997Co-Authors: Ian Stansfield, Vitaly V Kushnirov, Kerrie M Jones, Mick F TuiteAbstract:Genetic studies have indicated that the product of the yeast SUP45 gene encodes a component of the translational-termination machinery. In higher eukaryotes, genes similar to SUP45 encode eukaryote release factor 1 (eRFI), which has a stop-codon-dependent peptidyl-release activity. Using a conditional-lethal mutant allele of SUP45 (sup4.5-2) and a combination of in vivo and in vitro approaches, we demonstrate that the product of the SUP45 gene (Sup45p or eRF1) is a factor required for translation termination in yeast. A homologous in vitro assay based on suppressor-tRNA-mediated readthrough of stop codons is used to show that a translating lysate from a sup45–2 mutant strain exhibits a termination defect when heated for short periods to greater than the non-permissive temperature (37°C). This defect can be complemented with a purified preparation of Sup45p (eRF1) expressed in Eschericha coli. The termination defect in this strain appears to be due to an inability of the Sup45p protein to bind the ribosome, resulting in vivo in a reduced ability of Sup45p to release nascent polypeptides from the ribosome at the non-permissive temperature. Cell-free translation lysates from the sup45-2 strain do not show a defect in sense-codon translation at the non-permissive temperature. These data demonstrate that yeast eRFl plays a role in translation termination and is functionally equivalent to its higher eukaryotic homologues.
Robert Tycko - One of the best experts on this subject based on the ideXlab platform.
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locating folds of the in register parallel β sheet of the Sup35p prion domain infectious amyloid
Proceedings of the National Academy of Sciences of the United States of America, 2014Co-Authors: Anton Gorkovskiy, Robert Tycko, Kent R Thurber, Reed B WicknerAbstract:The [PSI+] prion is a self-propagating amyloid of the translation termination factor, Sup35p, of Saccharomyces cerevisiae. The N-terminal 253 residues (NM) of this 685-residue protein normally function in regulating mRNA turnover but spontaneously form infectious amyloid in vitro. We converted the three Ile residues in Sup35NM to Leu and then replaced 16 single residues with Ile, one by one, and prepared Ile-1-(13)C amyloid of each mutant, seeding with amyloid formed by the reference sequence Sup35NM. Using solid-state NMR, we showed that 10 of the residues examined, including six between residues 30 and 90, showed the ∼0.5-nm distance between labels diagnostic of the in-register parallel amyloid architecture. The five scattered N domain residues with wider spacing may be in turns or loops; one is a control at the C terminus of M. All mutants, except Q56I, showed little or no [PSI+] transmission barrier from the reference sequence, suggesting that they could assume a similar amyloid architecture in vitro when seeded with filaments of reference sequence Sup35NM. Infection of yeast cells expressing the reference SUP35 gene sequence with amyloid of several mutants produced [PSI+] transfectants with similar efficiency as did reference sequence Sup35NM amyloid. Our work provides a stringent demonstration that the Sup35 prion domain has the folded in-register parallel β-sheet architecture and suggests common locations of the folds. This architecture naturally suggests a mechanism of inheritance of conformation, the central mystery of prions.
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two prion variants of Sup35p have in register parallel β sheet structures independent of hydration
Biochemistry, 2009Co-Authors: Frank Shewmaker, Robert Tycko, Dmitry Kryndushkin, Bo Chen, Reed B WicknerAbstract:The [PSI+] prion is a self-propagating amyloid of the Sup35 protein, normally a subunit of the translation termination factor, but impaired in this vital function when in the amyloid form. The Sup35 N, M, and C domains are the amino-terminal prion domain, a connecting polar domain, and the essential C-terminal domain resembling eukaryotic elongation factor 1α respectively. Different [PSI+] isolates (prion variants) may have distinct biological properties, associated with different amyloid structures. Here we use solid state NMR to examine the structure of infectious Sup35NM amyloid fibrils of two prion variants. We find that both variants have an in-register parallel β-sheet structure, both in the fully hydrated form and in the lyophilized form. Moreover, we confirm that some leucine residues in the M domain participate in the in-register parallel β-sheet structure. Transmission of the [PSI+] prion by amyloid fibrils of Sup35NM and transmission of the [URE3] prion by amyloid fibrils of recombinant full-le...
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amyloid of rnq1p the basis of the pin prion has a parallel in register β sheet structure
Proceedings of the National Academy of Sciences of the United States of America, 2008Co-Authors: Reed B Wickner, Fred Dyda, Robert TyckoAbstract:The [PIN+] prion, a self-propagating amyloid form of Rnq1p, increases the frequency with which the [PSI+] or [URE3] prions arise de novo. Like the prion domains of Sup35p and Ure2p, Rnq1p is rich in N and Q residues, but rnq1Δ strains have no known phenotype except for inability to propagate the [PIN+] prion. We used solid-state NMR methods to examine amyloid formed in vitro from recombinant Rnq1 prion domain (residues 153–405) labeled with Tyr-1–13C (14 residues), Leu-1–13C (7 residues), or Ala-3–13C (13 residues). The carbonyl chemical shifts indicate that most Tyr and Leu residues are in β-sheet conformation. Experiments designed to measure the distance from each labeled residue to the next nearest labeled carbonyl showed that almost all Tyr and Leu carbonyl carbon atoms were ≈0.5 nm from the next nearest Tyr and Leu residues, respectively. This result indicates that the Rnq1 prion domain forms amyloid consisting of parallel β-strands that are either in register or are at most one amino acid out of register. Similar experiments with Ala-3–13C indicate that the β-strands are indeed in-register. The parallel in-register structure, now demonstrated for each of the yeast prions, explains the faithful templating of prion strains, and suggests as well a mechanism for the rare hetero-priming that is [PIN+]'s defining characteristic.
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amyloid of the prion domain of Sup35p has an in register parallel β sheet structure
Proceedings of the National Academy of Sciences of the United States of America, 2006Co-Authors: Frank Shewmaker, Reed B Wickner, Robert TyckoAbstract:The [PSI+] prion of Saccharomyces cerevisiae is a self-propagating amyloid form of Sup35p, a subunit of the translation termination factor. Using solid-state NMR we have examined the structure of amyloid fibrils formed in vitro from purified recombinant Sup351–253, consisting of the glutamine- and asparagine-rich N-terminal 123-residue prion domain (N) and the adjacent 130-residue highly charged M domain. Measurements of magnetic dipole–dipole couplings among 13C nuclei in a series of Sup35NM fibril samples, 13C-labeled at backbone carbonyl sites of Tyr, Leu, or Phe residues or at side-chain methyl sites of Ala residues, indicate intermolecular 13C–13C distances of ≈0.5 nm for nearly all sites in the N domain. Certain sites in the M domain also exhibit intermolecular distances of ≈0.5 nm. These results indicate that an in-register parallel β-sheet structure underlies the [PSI+] prion phenomenon.