DOCSLIB.ORG

Is a Lethal Yeast Prion

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Is a Lethal Yeast Prion Suicidal [PSI+] is a lethal yeast prion Ryan P. McGlinchey, Dmitry Kryndushkin, and Reed B. Wickner1 Laboratory of Biochemistry and Genetics, National Institute of Diabetes Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892-0830 Contributed by Reed B. Wickner, February 17, 2011 (sent for review January 28, 2011) [PSI+] is a prion of the essential translation termination factor codons in ade1-14 or ade2-1 is then sufficiently frequent that the Sup35p. Although mammalian prion infections are uniformly fatal, cells are Ade+ (grow without adenine and are white) instead of − commonly studied [PSI+] variants do not impair growth, leading to Ade (and red). A [PSI+] variant that efficiently inactivated all suggestions that [PSI+] may protect against stress conditions. We Sup35p (“killer [PSI+]”) would be lethal. As a permissive condi- report here that over half of [PSI+] variants are sick or lethal. These tion for killer [PSI+], we express a full-length chromosomal “killer [PSI+]s” are compatible with cell growth only when also SUP35 gene, and, on a plasmid, SUP35C encoding only the es- expressing minimal Sup35C, lacking the N-terminal prion domain. sential part but lacking the NM prion domain. This truncated The severe detriment of killer [PSI+] results in rapid selection of Sup35C cannot be converted to the [PSI+] prion form because it nonkiller [PSI+] variants or loss of the prion. We also report variants lacks the N domain that is essential for prion formation (18) and of [URE3], a prion of the nitrogen regulation protein Ure2p, that forms the core of the in-register parallel β-sheet amyloid structure grow much slower than ure2Δ cells. Our findings give a more re- that constitutes the prion form (19, 20). To detect killer [PSI+], alistic picture of the impact of the prion change than does focus on the plasmid-borne SUP35C must be expressed only enough to “mild” prion variants. keep cells alive but not enough for efficient termination, which − would make them Ade (Fig. S1). With a tetracycline-repressible rep lthough mammalian prions cause a devastating and uniformly promoter (Ptet ) (21), 10 μg/mL doxycycline sufficiently re- Afatal spongiform encephalopathy (reviewed in ref. 1), yeast pressed the plasmid-encoded Sup35C expression that cells lack- + and fungal prions, as described so far, are compatible with in- ing a chromosomal SUP35 were Ade (white) but could grow fi slowly (Fig. S2). Doxycycline at these levels does not detectably de nite growth, often at normal or near-normal rates. This ap- GENETICS rep parent contrast has led to proposals that the yeast and fungal slow growth of normal cells. The tet -SUP35C gene was on prions may benefit their hosts, even though prion formation may a URA3 CEN (centromere-containing) vector whose loss could be decrease the activity of the protein as a result of the aggregate/ selected by growth on plates with 5-fluorouracil (FOA; kills URA3 amyloid formation that is the prion change. The [Het-s] prion of cells but not ura3 mutants) (22). Podospora anserina is necessary for proper heterokaryon incom- Using strains 74-D694 or GT159 carrying PGAL1-SUP35NM rep + patibility of het-s with het-S strains (2), so it was proposed that this and URA3 Ptet -SUP35C plasmids, we induced [PSI ] forma- is a prion benefiting the host (3). However, [Het-s] is also part of tion by overexpression of Sup35NM on galactose media and a meiotic drive system, promoting inheritance of het-s by cheating selected Ade+ clones on plates with 10 μg/mL doxycycline. on meiosis (4), so the beneficial prion suggestion may be wrong. Artificially overproducing Sup35NM (the prion domain) can [PSI+] is an amyloid prion of the Saccharomyces cerevisiae Sup35p produce lethality because all Sup35p is drawn into the filaments (5–7), and was suggested to protect against stress (8) or to promote (23); thus, we repressed expression of Sup35NM by over 30 evolution by protecting against stress (9). All these tests of stress generations of growth on glucose to dilute out the Sup35NM resistance were done using the usual [PSI+] variants, which were before looking for colonies that grew poorly or not at all on FOA selected to be compatible with vigorous cell growth (10). medium (loss of Sup35C). Sup35p is an essential subunit, with Sup45p, of the translation We found that of 313 Ade+ guanidine-curable clones from termination factor. Like the mammalian prion “strains” (11, 12), 74-D694, 144 showed slow growth on FOA (“sick [PSI+]”) and [PSI+] has “variants,” several different stably propagated biolo- 24 did not grow at all (killer [PSI+]) (Fig. 1). Similar proportions gically and structurally different forms of the same prion protein of sick and killer [PSI+] were isolated from strain GT159 (Fig. (13, 14). In the usual [PSI+] variants, much of the Sup35p is tied S3). Cells with killer [PSI+] grew very poorly after several days up in amyloid filaments and is unavailable for translation ter- on ½YPD containing 10 μg/mL doxycycline, confirming that mination, but enough is free to keep the cells alive. The shortage failure to grow on FOA is attributable to lack of Sup35C (Fig. of Sup35p increases the misreading of nonsense codons, which 2A) and not, for example, a variant that never loses the URA3 provides the basis for the genetic assay of [PSI+]. plasmid. Most of the colonies that did grow were pink/red and Ure2p is a regulator of nitrogen catabolism that can convert to proved to have lost [PSI+]. On ½YPD lacking doxycycline, killer an amyloid prion called [URE3] (5, 15, 16). Variants of [URE3] [PSI+] cells were red and grew well because of an abundant have also been described (16, 17). supply of Sup35C (Fig. 2B). Here, we show that more than half of [PSI+] variants are lethal The instability of sick and killer [PSI+] is shown in Fig. 3. or highly pathogenic, unlike the variants usually studied. Simi- [PSI+] isolates were streaked on both FOA (Fig. 3A, Left) and larly, we describe abundant [URE3] variants that slow cell ½YPD without doxycycline (Fig. 3A, Center). After several days growth dramatically in a background in which deletion of the on ½YPD, sick and killer cells grew more normally when URE2 gene has no effect on growth. These results show that streaked to FOA (Fig. 3A, Right), showing that toxic [PSI+] acquisition of a yeast prion may be disastrous for yeast. variants are not stable, even in the presence of excess Sup35C. This suggests that toxicity is not limited to undersupply of active Results Sup35p has two domains, the N-terminal (NM) domain, which is necessary and sufficient for prion formation (and has physiolog- Author contributions: R.P.M., D.K., and R.B.W. designed research, performed research, ical function as well, as discussed below) but is dispensable for contributed new reagents/analytic tools, and wrote the paper. growth, and the C-terminal C domain, which is essential for The authors declare no conflict of interest. translation termination, and can perform this function without 1To whom correspondence should be addressed. E-mail: [email protected]. + fi the NM domain. In [PSI ] cells, most of the Sup35p is in la- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. ments, and is inactive. Readthrough of premature termination 1073/pnas.1102762108/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1102762108 PNAS Early Edition | 1of5 Downloaded by guest on September 23, 2021 A Single colony 5-FOA ½ YPD 5-FOA from ½ YPD Non-toxic PSI+ Sick PSI+ Sick PSI+ Killer PSI+ Fig. 1. Screening of Ade+ colonies for killer [PSI+]. Isolated Ade+ colonies Killer PSI+ were restreaked three times on −Ade −Ura + 10 μg/mL doxycycline before being stamped on FOA. Circled colonies have killer [PSI+]. The fractions of PSI+ ½ YPD from 5-FOA isolates with mild, sick, and killer [ ] are shown below. B C D Non-toxic Sick PSI+ Sick PSI+ PSI+ Sup35p and/or that the toxic [PSI+] variants are inherently un- stable. Streaking sick [PSI+] isolates on ½YPD after selection on − FOA showed a mixture of small white (Ade+ Ura ) and large − − pink/red (Ade Ura ) colonies (Fig. 3B, Upper). These small, white, poorly growing colonies, on restreaking on ½YPD, formed a mixture of small and larger white Ade+ colonies and pink/red − Ade colonies, with the latter having lost [PSI+] (Fig. 3B, Lower). Thus, selection for prion loss or alteration attributable to prion toxicity, and likely inherent prion instability, makes isola- + tion of stable killer [PSI ] apparently impossible. ½YPD ½YPD ½YPD Growth of sick [PSI+] isolates with 3 mM guanidine (which PSI+ A PSI+ cures [PSI+]) (24) cured the prion, as shown by cells becoming Fig. 3. Toxicity and instability of [ ] isolates. ( ) Sick and killer [ ] − − − μ candidates were streaked on ½YPD and on FOA plates. After 2 d of growth Ade on the Ade Ura + 10 g/mL doxycycline plates. Once B–D PSI+ + on ½YPD, strains were restreaked on FOA plates. ( ) Individual sick [ ] cured of killer [PSI ], these cells could grow well on FOA plates and nontoxic [PSI+] colonies were streaked on ½YPD from FOA. (Lower) or on ½YPD with doxycycline, similar to those shown in Fig. 3A. Single colonies restreaked on ½YPD are indicated. This shows that the toxicity is indeed attributable to a variant of [PSI+]. Transformation of presumed [PSI+] cells with a plasmid expressing Sup35NM-GFP confirmed the presence of prion color of yeast colonies (16). Strain BY241 was transformed with aggregates (Fig. 4). An apparent correlation between [PSI+] the LEU2 centromeric plasmid pVTG12 expressing Ure2N-GFP toxicity and the number of Sup35NM-GFP aggregates was noted (a fusion of the Ure2p prion domain with GFP) under control (Fig.
Load more

Recommended publications
  • Variable Absorption of Mutational Trends by Prion-Forming Domains During Saccharomycetes Evolution
    Variable absorption of mutational trends by prion-forming domains during Saccharomycetes evolution Paul M. Harrison Department of Biology, McGill University, Monteal, Quebec, Canada ABSTRACT Prions are self-propagating alternative states of protein domains. They are linked to both diseases and functional protein roles in eukaryotes. Prion-forming domains in Saccharomyces cerevisiae are typically domains with high intrinsic protein disorder (i.e., that remain unfolded in the cell during at least some part of their functioning), that are converted to self-replicating amyloid forms. S. cerevisiae is a member of the fungal class Saccharomycetes, during the evolution of which a large population of prion-like domains has appeared. It is still unclear what principles might govern the molecular evolution of prion-forming domains, and intrinsically disordered domains generally. Here, it is discovered that in a set of such prion-forming domains some evolve in the fungal class Saccharomycetes in such a way as to absorb general mutation biases across millions of years, whereas others do not, indicating a spectrum of selection pressures on composition and sequence. Thus, if the bias-absorbing prion formers are conserving a prion-forming capability, then this capability is not interfered with by the absorption of bias changes over the duration of evolutionary epochs. Evidence is discovered for selective constraint against the occurrence of lysine residues (which likely disrupt prion formation) in S. cerevisiae prion-forming domains as they evolve across Saccharomycetes. These results provide a case study of the absorption of mutational trends by compositionally biased domains, and suggest methodology for assessing selection pressures on the composition of intrinsically disordered regions.
    [Show full text]
  • Sporadic Distribution of Prion-Forming Ability of Sup35p from Yeasts and Fungi
    HIGHLIGHTED ARTICLE INVESTIGATION Sporadic Distribution of Prion-Forming Ability of Sup35p from Yeasts and Fungi Herman K. Edskes,1 Hima J. Khamar, Chia-Lin Winchester, Alexandria J. Greenler, Albert Zhou, Ryan P. McGlinchey, Anton Gorkovskiy, and Reed B. Wickner Laboratory of Biochemistry and Genetics, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892-0830 ABSTRACT 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,andKluyveromyces 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,orCryptococcus neoformans.EachoftwoC. 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.
    [Show full text]
  • In Saccharomyces Cerevisiae by Environmental Stress Through a Prion-Mediated Mechanism
    The EMBO Journal Vol.18 No.7 pp.1974–1981, 1999 Translation termination efficiency can be regulated in Saccharomyces cerevisiae by environmental stress through a prion-mediated mechanism Simon S.Eaglestone, Brian S.Cox and In yeast, the [PSI1] factor is a product of the SUP35 Mick F.Tuite1 gene (Chernoff et al., 1993; Doel et al., 1994; Ter- Avanesyan et al., 1994) which encodes eRF3 (Sup35p), an Research School of Biosciences, University of Kent, Canterbury, essential eukaryotic polypeptide release factor. Eukaryote Kent CT2 7NJ, UK translation termination is mediated by a soluble cyto- 1Corresponding author plasmic complex, which encompasses eRF3 and at least e-mail: [email protected] one other factor, namely eRF1 [Sup45p] (Stansfield et al., 1995a; Zhouravleva et al., 1995). As well as folding into F [PSI ] is a protein-based heritable phenotype of the its native structure, Sup35p is believed to be capable of yeast Saccharomyces cerevisiae which reflects the prion- adopting a second aberrant conformation, which manifests like behaviour of the endogenous Sup35p protein F as the prion-associated phenotype (Chernoff et al., 1995; release factor. [PSI ] strains exhibit a marked Paushkin et al., 1996; Tuite and Lindquist, 1996). In decrease in translation termination efficiency, which [PSI1] strains, Sup35p is present both as a soluble factor permits decoding of translation termination signals and as large intracellular aggregates, resulting from the and, presumably, the production of abnormally propensity of the prion conformer to coalesce (Patino et al., extended polypeptides. We have examined whether the F 1996; Paushkin et al., 1996). The resulting intracellular [PSI ]-induced expression of such an altered proteome depletion of soluble termination factors facilitates the might confer some selective growth advantage over decoding of termination signals by mutant nonsense [psi–] strains.
    [Show full text]
  • Ribosomal RNA Modulates Aggregation of the Podospora Prion Protein HET-S
    International Journal of Molecular Sciences Article Ribosomal RNA Modulates Aggregation of the Podospora Prion Protein HET-s Yanhong Pang y, Petar Kovachev and Suparna Sanyal * Department of Cell and Molecular Biology, Uppsala University, Box-596, Biomedical Center, 751 24 Uppsala, Sweden; [email protected] (Y.P.); [email protected] (P.K.) * Correspondence: [email protected] Present address: Department of Molecular Sciences, Uppsala Biocenter, Swedish University of Agricultural y Sciences, 750 07 Uppsala, Sweden. Received: 25 August 2020; Accepted: 28 August 2020; Published: 1 September 2020 Abstract: The role of the nucleic acids in prion aggregation/disaggregation is becoming more and more evident. Here, using HET-s prion from fungi Podospora anserina (P. anserina) as a model system, we studied the role of RNA, particularly of different domains of the ribosomal RNA (rRNA), in its aggregation process. Our results using Rayleigh light scattering, Thioflavin T (ThT) binding, transmission electron microscopy (TEM) and cross-seeding assay show that rRNA, in particular the domain V of the major rRNA from the large subunit of the ribosome, substantially prevents insoluble amyloid and amorphous aggregation of the HET-s prion in a concentration-dependent manner. Instead, it facilitates the formation of the soluble oligomeric “seeds”, which are capable of promoting de novo HET-s aggregation. The sites of interactions of the HET-s prion protein on domain V rRNA were identified by primer extension analysis followed by UV-crosslinking, which overlap with the sites previously identified for the protein-folding activity of the ribosome (PFAR). This study clarifies a missing link between the rRNA-based PFAR and the mode of propagation of the fungal prions.
    [Show full text]
  • Regulation of Synaptic Pumilio Function by an Aggregation-Prone Domain
    The Journal of Neuroscience, January 13, 2010 • 30(2):515–522 • 515 Development/Plasticity/Repair Regulation of Synaptic Pumilio Function by an Aggregation-Prone Domain Anna M. Salazar, Edward J. Silverman, Kaushiki P. Menon, and Kai Zinn Division of Biology, California Institute of Technology, Pasadena, California 91125 We identified Pumilio (Pum), a Drosophila translational repressor, in a computational search for metazoan proteins whose activities mightberegulatedbyassemblyintoorderedaggregates.Thesearchalgorithmwasbasedonevolutionarysequenceconservationpatterns observed for yeast prion proteins, which contain aggregation-prone glutamine/asparagine (Q/N)-rich domains attached to functional domains of normal amino acid composition. We examined aggregation of Pum and its nematode ortholog PUF-9 by expression in yeast. A domain of Pum containing the Q/N-rich sequence, denoted as NQ1, the entire Pum N terminus, and the complete PUF-9 protein localize .to macroscopic aggregates (foci) in yeast. NQ1 and PUF-9 can generate the yeast Pin؉ trait, which is transmitted by a heritable aggregate NQ1 also assembles into amyloid fibrils in vitro.InDrosophila, Pum regulates postsynaptic translation at neuromuscular junctions (NMJs). To assess whether NQ1 affects synaptic Pum activity in vivo, we expressed it in muscles. We found that it negatively regulates endogenous Pum, producing gene dosage-dependent pum loss-of-function NMJ phenotypes. NQ1 coexpression also suppresses lethality andNMJphenotypescausedbyoverexpressionofPuminmuscles.TheQ/NblockofNQ1isrequiredforthesephenotypiceffects.Negative regulation of Pum by NQ1 might be explained by formation of inactive aggregates, but we have been unable to demonstrate that NQ1 aggregates in Drosophila. NQ1 could also regulate Pum by a “dominant-negative” effect, in which it would block Q/N-mediated interac- tions of Pum with itself or with cofactors required for translational repression.
    [Show full text]
  • [PSI+]: an Epigenetic Modulator of Translation Termination Efficiency, 661 Tricia R
    P1: FHR/fob P2: FHN-FKP/FGO QC: FKP September 9, 1999 15:50 Annual Reviews AR092-21 Annu. Rev. Cell Dev. Biol. 1999. 15:661–703 Copyright c 1999 by Annual Reviews. All rights reserved [PSI +]: An Epigenetic Modulator of Translation Termination Efficiency # Tricia R. Serio and Susan L. Lindquist The University of Chicago, Department of Molecular Genetics and Cell Biology, #Howard Hughes Medical Institute, Chicago, Illinois 60637; e-mail: [email protected]; [email protected] Key Words amyloid, prion, Sup35, nonsense suppression, epigenetic ■ Abstract The [PSI+] factor of the yeast Saccharomyces cerevisiae is an epige- netic regulator of translation termination. More than three decades ago, genetic analy- sis of the transmission of [PSI+] revealed a complex and often contradictory series of observations. However, many of these discrepancies may now be reconciled by a rev- olutionary hypothesis: protein conformation-based inheritance (the prion hypothesis). This model predicts that a single protein can stably exist in at least two distinct physical states, each associated with a different phenotype. Propagation of one of these traits is achieved by a self-perpetuating change in the protein from one form to the other. Mounting genetic and biochemical evidence suggests that the determinant of [PSI+] is the nuclear encoded Sup35p, a component of the translation termination complex. Here we review the series of experiments supporting the yeast prion hypothesis and provide another look at the 30 years of work preceding this theory in light of our current state of knowledge. CONTENTS Introduction .................................................... 662 The Phenomenon of Nonsense Suppression...........................663 by Massachusetts Institute of Technology (MIT) on 06/18/13.
    [Show full text]
  • [41] Yeast Prion [~÷] and Its Determinant, Sup35p ANTHONY S. KOWAL, and SUSAN L. LINDOUIST Introduction [PS/+] and [URE3] Are T
    [411 YEAST PRION [XIY+] AND ITS DETERMINANT, Sup35p 649 [41] Yeast Prion [~÷] and Its Determinant, Sup35p By TRICIA R. SERIO, ANIL G. CASHIKAR, JAHAN J. MOSLEHI, ANTHONY S. KOWAL, and SUSAN L. LINDOUIST Introduction [PS/+] and [URE3] are two non-Mendelian genetic elements of the yeast Saccharomyces cerevisiae that appear to be inherited through an unusual mechanism: the continued propagation of an alternate protein conformation. The protein determinants of these elements, Sup35p for [PSI+] 1"2 and Ure2p for [URE3], 3'4 have the unique ability to exist in at least two different, stable conformations in vivo. 4-8 Although the spontane- ous generation of one conformer is rare, this alternate form, once ac- quired, becomes predominant, influencing the other conformer to change states. 5 This self-perpetuation of protein conformation is the key to the non-Mendelian inheritance of both [PSI +] and [URE3]. In addition, the [Het-S] phenotype of Podospora anserina, another fungus, may be inherited by a similar mechanism.9 This article focuses on both in vivo and in vitro methods used to analyze [PSI+], the most extensively studied member of this group. Genetics of [PSI +] Inheritance [PSI +] was originally described in 1965 by Cox as a translation infidelity factor. I° Translation terminated efficiently at nonsense codons in strains classified as [psi-], whereas [PSI +] strains were capable of omnipotently 1 S. M. Doel, S. J. McCready, C. R. Nierras, and B. S. Cox, Genetics 137, 659 (1994). 2 M. D. Ter-Avanesyan, A. R. Dagkesamanskaya, V. V. Kushnirov, and V. N. Smirnov, Genetics 137, 671 (1994). 3 R.
    [Show full text]
  • Normal Levels of Ribosome-Associated Chaperones Cure Two Groups of [PSI+] Prion Variants
    Normal levels of ribosome-associated chaperones cure two groups of [PSI+] prion variants Moonil Sona and Reed B. Wicknera,1 aLaboratory of Biochemistry and Genetics, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892-0830 Contributed by Reed B. Wickner, September 9, 2020 (sent for review August 11, 2020; reviewed by Chih-Yen King and Susan Liebman) The yeast prion [PSI+] is a self-propagating amyloid of the trans- prions (24), and in the curing of [PSI+] by its overproduction (23). lation termination factor, Sup35p. For known pathogenic prions, These two different activities of Hsp104 were then separated by such as [PSI+], a single protein can form an array of different am- showing that disruption of the Hsp104 N-terminal region elimi- yloid structures (prion variants) each stably inherited and with nates the Hsp104 overproduction curing ability without any effect differing biological properties. The ribosome-associated chaper- on [PSI+] propagation (25). In fact, spontaneous [PSI+] gener- ones, Ssb1/2p (Hsp70s), and RAC (Zuo1p (Hsp40) and Ssz1p ation was elevated by over 10-fold in an N-terminal mutant, T160M (Hsp70)), enhance de novo protein folding by protecting nascent hsp104 , and the normal level of WT Hsp104 was able to cure polypeptide chains from misfolding and maintain translational many of the [PSI+] variants arising in the mutant strain (26). fidelity by involvement in translation termination. Ssb1/2p and Overproduced endosomal sorting factor Btn2p and its paralo- RAC chaperones were previously found to inhibit [PSI+] prion gen- gue Cur1p were shown to cure the [URE3] prion, in the case of eration.
    [Show full text]
  • The Strain of Being a Prion Marston Exhibited a Number of Flints Which Mick F
    news and views To achieve the necessary sharper imaging, They show absorption spectra — corre- astronomers have resorted to a ‘shadow- sponding to their radiation being absorbed gram’technique,using a metal mask encoded by some intervening material between these with a pattern of holes to cast a shadow on a sources and INTEGRAL, meaning that large camera (Fig. 1). By correlating the they stand out only at ȍ-ray wavelengths. observed shadow with the known pattern in The absorbing material may be either the the coded mask, the position of the ȍ-ray accretion flow from a companion star, or a source can be pinpointed.The viability of the molecular cloud in which the source is 100 YEARS AGO technique was demonstrated by a pathfinder embedded. Now we know where these I happened to possess a small sample of a Russian–French telescope that flew on the objects are, follow-up observations using red deposit, coloured by iron, which is left by Russian GRANAT mission in 1989. other telescopes, such as ESA’s XMM– the water of the King’s Spring, at Bath. It Coded-mask telescopes need large Newton Observatory, will uncover their occurred to me that it might be worth while radiation-collecting areas to detect the faint nature more fully. to test this for radio-activity. The result was sources,and this in turn requires a big,heavy Over the coming years, INTEGRAL will to show that the deposit was markedly space observatory. For more than two make a complete census of the buried popu- active. On leaving it in the testing vessel decades, astronomers could only dream of lations of black holes and neutron stars in the (which was closed airtight) for a few days, the potential of such a ȍ-ray telescope for Milky Way.
    [Show full text]
  • Discovery and Characterization of Prions in Saccharomyces Cerevisiae
    Discovery and Characterization of Prions in Saccharomyces cerevisiae by Randal A. Halfmann B.S. Genetics Texas A&M University, 2004 Submitted to the Department of Biology in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY IN BIOLOGY at the MASSACHUSETTS INSTITUTE OF TECHNOLOGY FEBRUARY 2011 © 2011 Randal A. Halfmann. All rights reserved. The author hereby grants to MIT permission to reproduce and to distribute publicly paper and electronic copies of this thesis document in whole or in part in any medium now known or hereafter created Signature of Author:_________________________________________________________________________________ Department of Biology December 3, 2010 Certified by:___________________________________________________________________________________________ Susan L. Lindquist Professor of Biology Thesis Supervisor Accepted by:__________________________________________________________________________________________ Stephen P. Bell Professor of Biology Co-Chair, Biology Graduate Committee 1 2 Discovery and Characterization of Prions in Saccharomyces cerevisiae by Randal A. Halfmann Submitted to the Department of Biology in partial fulfillment of the requirements for the Degree of Doctor of Philosophy in Biology ABSTRACT Some protein aggregates can perpetuate themselves in a self-templating protein-misfolding reaction. These aggregates, or prions, are the infectious agents behind diseases like Kuru and mad- cow disease. In yeast, however, prions act as epigenetic elements that confer heritable alternative phenotypes. Prion-forming proteins create bistable molecular systems whose semi-stochastic switching between functional states increases phenotypic diversity within cell populations. My thesis work explores the idea that rather than being detrimental, prions may commonly act to their host’s advantage. To broaden the known range of prion phenomena in S. cerevisiae, I, together with a postdoctoral fellow in our lab, systematically surveyed the yeast proteome for prion-forming proteins.
    [Show full text]
  • The Strength of Selection Against the Yeast Prion [PSI ]
    Genetics: Published Articles Ahead of Print, published on February 25, 2009 as 10.1534/genetics.108.100297 1 The strength of selection against the yeast prion [PSI+] Joanna Masel and Cortland K. Griswold1 Dpt. Ecology & Evolutionary Biology, University of Arizona, Tucson, AZ, 85721, USA. 1present address: Dpt. Integrative Biology, University of Guelph, Guelph, Ontario, N1G 2W1, Canada. 2 Running heading: Selection against the [PSI+] prion Keywords: population genetics, readthrough translation, nonsense suppression, inbreeding, indirect selection Joanna Masel Dpt. Ecology & Evolutionary Biology 1041 E. Lowell St. University of Arizona Tucson, AZ 85721 Phone: 520-626-9888 [email protected] 3 Abstract The [PSI+] prion causes widespread readthrough translation and is rare in natural populations of Saccharomyces, despite the fact that sex is expected to cause it to spread. Using the recently estimated rate of Saccharomyces outcrossing, we calculate the strength of selection necessary to maintain [PSI+] at levels low enough to be compatible with data. Using the best available parameter estimates, we find selection against [PSI+] to be significant. Inference regarding selection on modifiers of [PSI+] appearance depends on obtaining more precise and accurate + estimates of the product of yeast effective population size Ne and the spontaneous rate of [PSI ] appearance m. The ability to form [PSI+] has persisted in yeast over a long period of evolutionary time, despite a diversity of modifiers that could abolish it. If mNe < 1, this may be explained by insufficiently strong selection. If mNe > 1, then selection should have favored the spread of [PSI+] resistance modifiers. In this case, rare conditions where [PSI+] is adaptive may permit its persistence in the face of negative selection.
    [Show full text]
  • Extracellular Vesicles-Encapsulated Yeast Prions and What They Can Tell Us About the Physical Nature of Propagons
    International Journal of Molecular Sciences Review Extracellular Vesicles-Encapsulated Yeast Prions and What They Can Tell Us about the Physical Nature of Propagons Mehdi Kabani Molecular Imaging Research Center (MIRCen), Laboratoire des Maladies Neurodégénératives (UMR9199), Université Paris-Saclay, CEA, CNRS, F-92265 Fontenay-aux-Roses, France; [email protected] Abstract: The yeast Saccharomyces cerevisiae hosts an ensemble of protein-based heritable traits, most of which result from the conversion of structurally and functionally diverse cytoplasmic proteins into prion forms. Among these, [PSI+], [URE3] and [PIN+] are the most well-documented prions and arise from the assembly of Sup35p, Ure2p and Rnq1p, respectively, into insoluble fibrillar assemblies. Yeast prions propagate by molecular chaperone-mediated fragmentation of these aggregates, which generates small self-templating seeds, or propagons. The exact molecular nature of propagons and how they are faithfully transmitted from mother to daughter cells despite spatial protein quality control are not fully understood. In [PSI+] cells, Sup35p forms detergent-resistant assemblies de- tectable on agarose gels under semi-denaturant conditions and cytosolic fluorescent puncta when the protein is fused to green fluorescent protein (GFP); yet, these macroscopic manifestations of [PSI+] do not fully correlate with the infectivity measured during growth by the mean of protein infection assays. We also discovered that significant amounts of infectious Sup35p particles are exported via extracellular (EV) and periplasmic (PV) vesicles in a growth phase and glucose-dependent manner. In the present review, I discuss how these vesicles may be a source of actual propagons and a suitable vehicle for their transmission to the bud. Keywords: yeast; prion; SUP35;[PSI+]; extracellular vesicles; exosomes; protein aggregation; protein quality control Citation: Kabani, M.
    [Show full text]