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Contributions of the Prion Protein Sequence, Strain, and Environment to the Species Barrier*

  • Aditi Sharma
    Correspondence
    Supported by the National Science Foundation - Industry/University Cooperative Research Center, Center for Pharmaceutical Development (National Science Foundation Grant 0969003). To whom correspondence may be addressed: School of Chemical and Biomolecular Engineering, Georgia Institute of Technology, Engineered Biosystems Building, M/C 2000, 950 Atlantic Dr., Atlanta, GA 30332–2000. Tel.: (404) 385-1334;
    Footnotes
    Affiliations
    From the Schools of Chemical & Biomolecular Engineering and
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  • Kathryn L. Bruce
    Footnotes
    Affiliations
    Biology, Georgia Institute of Technology, Atlanta, Georgia 30332 and
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  • Buxin Chen
    Footnotes
    Affiliations
    Biology, Georgia Institute of Technology, Atlanta, Georgia 30332 and
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  • Stefka Gyoneva
    Footnotes
    Affiliations
    Biology, Georgia Institute of Technology, Atlanta, Georgia 30332 and
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  • Sven H. Behrens
    Affiliations
    From the Schools of Chemical & Biomolecular Engineering and
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  • Andreas S. Bommarius
    Correspondence
    Supported by the National Science Foundation - Industry/University Cooperative Research Center, Center for Pharmaceutical Development (National Science Foundation Grant 0969003). To whom correspondence may be addressed: School of Chemical and Biomolecular Engineering, Georgia Institute of Technology, Engineered Biosystems Building, M/C 2000, 950 Atlantic Dr., Atlanta, GA 30332–2000. Tel.: (404) 385-1334;
    Affiliations
    From the Schools of Chemical & Biomolecular Engineering and
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  • Yury O. Chernoff
    Correspondence
    Supported by Russian Science Foundation Grant 14-50-00069 and Russian Foundation for Basic Research Grant 15-04-06650. To whom correspondence may be addressed: School of Biology, Georgia Institute of Technology, Engineered Biosystems Building, M/C 2000, 950 Atlantic Dr., Atlanta, GA 30332-2000. Tel.: 404-894-1157; Fax: 404-894-0519;
    Affiliations
    Biology, Georgia Institute of Technology, Atlanta, Georgia 30332 and

    the Laboratory of Amyloid Biology and Institute of Translational Biomedicine, St. Petersburg State University, St. Petersburg 199034, Russia
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  • Author Footnotes
    * The authors declare that they have no conflicts of interest with the contents of this article.
    This article contains supplemental Figures S1 and S2.
    8 K. L. Bruce, B. Chen, S. Gyoneva, and Y. O. Chernoff, unpublished data.
    1 Both authors contributed equally to this work.
    3 Supported by a Graduate Assistance in the Areas of National Need fellowship from the U.S. Department of Education.
    4 Present address: COI Pharmaceuticals Inc., 11099 N. Torrey Pines Rd., Ste. 290, La Jolla, CA 92037.
    5 Supported by a Petite undergraduate research scholarship from the Institute for Bioengineering and Bioscience. Present address: Biogen, 225 Binney St., Cambridge, MA 02142.
Open AccessPublished:November 12, 2015DOI:https://doi.org/10.1074/jbc.M115.684100
      Amyloid propagation requires high levels of sequence specificity so that only molecules with very high sequence identity can form cross-β-sheet structures of sufficient stringency for incorporation into the amyloid fibril. This sequence specificity presents a barrier to the transmission of prions between two species with divergent sequences, termed a species barrier. Here we study the relative effects of protein sequence, seed conformation, and environment on the species barrier strength and specificity for the yeast prion protein Sup35p from three closely related species of the Saccharomyces sensu stricto group; namely, Saccharomyces cerevisiae, Saccharomyces bayanus, and Saccharomyces paradoxus. Through in vivo plasmid shuffle experiments, we show that the major characteristics of the transmission barrier and conformational fidelity are determined by the protein sequence rather than by the cellular environment. In vitro data confirm that the kinetics and structural preferences of aggregation of the S. paradoxus and S. bayanus proteins are influenced by anions in accordance with their positions in the Hofmeister series, as observed previously for S. cerevisiae. However, the specificity of the species barrier is primarily affected by the sequence and the type of anion present during the formation of the initial seed, whereas anions present during the seeded aggregation process typically influence kinetics rather than the specificity of prion conversion. Therefore, our work shows that the protein sequence and the conformation variant (strain) of the prion seed are the primary determinants of cross-species prion specificity both in vivo and in vitro.

      Introduction

      Amyloidogenic proteins form ordered self-seeding fibrous aggregates that are known to be associated with a variety of neurodegenerative diseases in humans and other mammals, such as Alzheimer disease, Parkinson disease, and prion diseases, including sheep scrapie, mad cow disease, elk and deer chronic wasting disease, and human Creutzfeldt-Jakob disease, kuru, and fatal familial insomnia (
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      Protein misfolding, functional amyloid, and human disease.
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      ). The amyloid form of an amyloidogenic protein can convert the cellular form of a protein of the same or very similar amino acid sequence into an amyloid conformation, usually via cross-β interactions (
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      ). Transmissible amyloids called prions can spread the amyloid state between organisms. Cross-species transmission of the prion state is impaired by sequence divergence within the prion proteins, resulting in a “species barrier” to transmission of a prion from one species to another (
      • Pattison I.H.
      Experiments with scrapie with special reference to the nature of the agent and the pathology of the disease: Slow, latent and temperate virus infections.
      ). However, the species barrier can be overcome in some species combinations. For example, bovine spongiform encephalopathy, which possibly originated from transmission of a scrapie prion from sheep to cattle, resulted in the widely known “mad cow” disease epidemic that greatly affected the United Kingdom in the 1990s (
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      ,
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      ). Bovine spongiform encephalopathy has also been found to be transmitted from cattle to humans, manifesting itself as a new variant of Creutzfeldt-Jakob disease called variant Creutzfeldt-Jakob disease (
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      Predicting the size of the epidemic of the new variant of Creutzfeldt-Jakob disease.
      ). Therefore, understanding the mechanisms of species barrier and cross-species prion transmission is crucial for preventing future outbreaks of prion diseases. However, the rules governing species barriers as well as the effects of the physiological and environmental conditions on the barrier are poorly understood to date.
      Yeast prions are cytoplasmic elements heritable in a non-Mendelian fashion (for a review, see Refs.
      • Liebman S.W.
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      Prions in yeast.
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      ). Because many of them control phenotypically detectable traits, yeast prions provide a useful model for studying the molecular basis of prion phenomena. One of the best studied yeast prion proteins is translation termination factor Sup35. Prion formation by Sup35 causes translational readthrough (nonsense suppression), a phenotypically detectable trait in specifically designed yeast strains (
      • Liebman S.W.
      • Chernoff Y.O.
      Prions in yeast.
      ,
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      Physiological and environmental control of yeast prions.
      ). Previously, we (
      • Chen B.
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      • Chernoff Y.O.
      Prion species barrier between the closely related yeast proteins is detected despite coaggregation.
      ,
      • Chen B.
      • Bruce K.L.
      • Newnam G.P.
      • Gyoneva S.
      • Romanyuk A.V.
      • Chernoff Y.O.
      Genetic and epigenetic control of the efficiency and fidelity of cross-species prion transmission.
      ) and others (
      • Afanasieva E.G.
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      • Ter-Avanesyan M.D.
      Molecular basis for transmission barrier and interference between closely related prion proteins in yeast.
      ) have reported observations of the species barrier and cross-species prion transmission between Sup35 proteins of three closely related yeast species, Saccharomyces cerevisiae, Saccharomyces paradoxus, and Saccharomyces bayanus, containing Sup35p with high levels of sequence similarity.
      The Sup35 protein can be divided into three segments: the N-terminal prion domain (Sup35N); the linker middle domain (Sup35M); and the functional C-terminal domain (Sup35C), responsible for translation termination and cell viability. Levels of similarity between the Sup35N fragments of the Sup35 proteins from Saccharomyces sensu stricto are close to those observed in mammalian prion proteins (
      • Wopfner F.
      • Weidenhöfer G.
      • Schneider R.
      • von Brunn A.
      • Gilch S.
      • Schwarz T.F.
      • Werner T.
      • Schätzl H.M.
      Analysis of 27 mammalian and 9 avian PrPs reveals high conservation of flexible regions of the prion protein.
      ,
      • Šandula J.
      • Vojtková-Lepšíková A.
      Immunochemical studies on mannans of the genus Saccharomyces.
      ), which renders this system a useful model for studying the species barrier. The Sup35N domain alone is difficult to express because it is poorly soluble and aggregates too rapidly, whereas the addition of the Sup35M domain resolves all of the abovementioned issues. Therefore, Sup35NM is used widely as a model protein for studying amyloid aggregation in vitro and has been shown to transmit prion properties to full-size Sup35 after transfection into yeast cells (
      • Tanaka M.
      • Chien P.
      • Naber N.
      • Cooke R.
      • Weissman J.S.
      Conformational variations in an infectious protein determine prion strain differences.
      ,
      • Tanaka M.
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      An efficient protein transformation protocol for introducing prions into yeast.
      ). The amino acid similarities between the Sup35NM regions of the three different species are 95% and 93% (S. cerevisiae/S. paradoxus combination), 85% and 78% (S. cerevisiae/S. bayanus combination), and 85% and 79% (S. paradoxus/S. bayanus combination) (
      • Chen B.
      • Newnam G.P.
      • Chernoff Y.O.
      Prion species barrier between the closely related yeast proteins is detected despite coaggregation.
      ,
      • Chen B.
      • Bruce K.L.
      • Newnam G.P.
      • Gyoneva S.
      • Romanyuk A.V.
      • Chernoff Y.O.
      Genetic and epigenetic control of the efficiency and fidelity of cross-species prion transmission.
      ). Our previous experiments, performed with divergent Sup35 proteins (or proteins containing divergent or chimeric Sup35N domains) in S. cerevisiae cells, have demonstrated that the prion species barrier depends on (but is not arithmetically proportional to) the level of divergence of Sup35N sequences and that different subregions (modules) of Sup35N play a primary role in determining the barrier in different cross-species combinations (
      • Chen B.
      • Newnam G.P.
      • Chernoff Y.O.
      Prion species barrier between the closely related yeast proteins is detected despite coaggregation.
      ,
      • Chen B.
      • Bruce K.L.
      • Newnam G.P.
      • Gyoneva S.
      • Romanyuk A.V.
      • Chernoff Y.O.
      Genetic and epigenetic control of the efficiency and fidelity of cross-species prion transmission.
      ,
      • Bruce K.L.
      • Chernoff Y.O.
      Sequence specificity and fidelity of prion transmission in yeast.
      ). A prion species barrier has also been observed with divergent Sup35NM fragments in vitro (
      • Chen B.
      • Newnam G.P.
      • Chernoff Y.O.
      Prion species barrier between the closely related yeast proteins is detected despite coaggregation.
      ).
      Prion proteins, including mammalian PrP and yeast Sup35, not only can fold into alternative prion and non-prion forms but can also adopt multiple distinct amyloid conformations, known as “strains” or “variants” (
      • Liebman S.W.
      • Chernoff Y.O.
      Prions in yeast.
      ,
      • Derkatch I.L.
      • Chernoff Y.O.
      • Kushnirov V.V.
      • Inge-Vechtomov S.G.
      • Liebman S.W.
      Genesis and variability of [PSI] prion factors in Saccharomyces cerevisiae.
      • Toyama B.H.
      • Kelly M.J.
      • Gross J.D.
      • Weissman J.S.
      The structural basis of yeast prion strain variants.
      ,
      • Stein K.C.
      • True H.L.
      Prion strains and amyloid polymorphism influence phenotypic variation.
      ,
      • Kretzschmar H.
      • Tatzelt J.
      Prion disease: a tale of folds and strains.
      ,
      • Parchi P.
      • Strammiello R.
      • Giese A.
      • Kretzschmar H.
      Phenotypic variability of sporadic human prion disease and its molecular basis: past, present, and future.
      ,
      • Gambetti P.
      • Cali I.
      • Notari S.
      • Kong Q.
      • Zou W.-Q.
      • Surewicz W.K.
      Molecular biology and pathology of prion strains in sporadic human prion diseases.
      • Surewicz W.K.
      • Apostol M.I.
      Prion protein and its conformational conversion: a structural perspective.
      ). Different strains are associated with distinct disease patterns in mammals and different stringencies of phenotypic effects in fungi (because of this, yeast prion strains can be termed “strong,” “intermediate,” “weak,” etc.). When formed, the prion strain is typically reproduced faithfully, although strain “mutations” may occur with a low frequency (
      • Bateman D.A.
      • Wickner R.B.
      [PSI+] prion transmission barriers protect Saccharomyces cerevisiae from infection: intraspecies ‘species barriers‘.
      • Bateman D.A.
      • Wickner R.B.
      The [PSI+] prion exists as a dynamic cloud of variants.
      ,
      • Li J.
      • Browning S.
      • Mahal S.P.
      • Oelschlegel A.M.
      • Weissmann C.
      Darwinian evolution of prions in cell culture.
      ,
      • Mahal S.P.
      • Browning S.
      • Li J.
      • Suponitsky-Kroyter I.
      • Weissmann C.
      Transfer of a prion strain to different hosts leads to emergence of strain variants.
      • Ghaemmaghami S.
      • Watts J.C.
      • Nguyen H.-O.
      • Hayashi S.
      • DeArmond S.J.
      • Prusiner S.B.
      Conformational transformation and selection of synthetic prion strains.
      ). In both mammals and yeast, prion strain properties influence the species barrier (
      • Bruce K.L.
      • Chernoff Y.O.
      Sequence specificity and fidelity of prion transmission in yeast.
      ,
      • Hill A.F.
      • Collinge J.
      ). For example, transmission of the specific strong prion strain from the S. cerevisiae to S. paradoxus Sup35 prion domain (PrD)
      The abbreviations used are: PrD, prion domain; Sc, Saccharomyces cerevisiae; Sp, Saccharomyces paradoxus; Sb, Saccharomyces bayanus; YPD, yeast extract/peptone/dextrose rich organic medium.
      in S. cerevisiae cells is relatively efficient, whereas transmission of the specific weak prion strain in the same direction is rare (
      • Chen B.
      • Bruce K.L.
      • Newnam G.P.
      • Gyoneva S.
      • Romanyuk A.V.
      • Chernoff Y.O.
      Genetic and epigenetic control of the efficiency and fidelity of cross-species prion transmission.
      ). Notably, underlying S. cerevisiae prion strain patterns were maintained during propagation through the S. paradoxus protein but altered irreversibly during propagation through the S. bayanus protein, which could be detected as a “switch” of prion strain after reverse transmission back to the S. cerevisiae sequence (
      • Chen B.
      • Bruce K.L.
      • Newnam G.P.
      • Gyoneva S.
      • Romanyuk A.V.
      • Chernoff Y.O.
      Genetic and epigenetic control of the efficiency and fidelity of cross-species prion transmission.
      ). This shows that not only the efficiency of prion transmission but also the fidelity of reproduction of prion conformation during transmission is controlled by the level of identity of interacting protein sequences.
      Previous work from our group has also demonstrated that the ion type present in solution greatly influences not only processes such as deactivation of enzymes (
      • Broering J.M.
      • Bommarius A.S.
      Evaluation of Hofmeister effects on the kinetic stability of proteins.
      ,
      • Broering J.M.
      • Bommarius A.S.
      Cation and strong co-solute effects on protein kinetic stability.
      • Broering J.M.
      • Bommarius A.S.
      Kinetic model for salt-induced protein deactivation.
      ) but also in vitro amyloid formation by S. cerevisiae Sup35NM and that ion effects are determined by their position in the Hofmeister series in the fashion of an “inverse” Hofmeister effect. Specifically, strongly hydrated anions (kosmotropes) initiate nucleation quickly and promote rapid fiber elongation, whereas poorly hydrated anions (chaotropes) delay nucleation and mildly affect the elongation rate (
      • Hofmeister F.
      On the understanding of the effects of salts.
      ,
      • Rubin J.
      • Khosravi H.
      • Bruce K.L.
      • Lydon M.E.
      • Behrens S.H.
      • Chernoff Y.O.
      • Bommarius A.S.
      Ion-specific effects on prion nucleation and strain formation.
      ). A similar effect of kosmotropes has also been observed by another group for the mammalian prion protein PrP (
      • Diaz-Espinoza R.
      • Mukherjee A.
      • Soto C.
      Kosmotropic anions promote conversion of recombinant prion protein into a PrPSc-like misfolded form.
      ). Moreover, amyloid formation by Sup35NM in the presence of different anions resulted in the generation of different spectra of prion strains, with kosmotropes favoring the formation of strong strains (characterized by smaller aggregate size and higher efficiency of fragmentation and proliferation), and chaotropes favoring the formation of weak strains (characterized by larger aggregate size and lower efficiency of fragmentation and proliferation) (
      • Hofmeister F.
      On the understanding of the effects of salts.
      ,
      • Rubin J.
      • Khosravi H.
      • Bruce K.L.
      • Lydon M.E.
      • Behrens S.H.
      • Chernoff Y.O.
      • Bommarius A.S.
      Ion-specific effects on prion nucleation and strain formation.
      ).
      In this work, we specifically address the contribution of the cellular composition and the conditions of the aggregation reaction to the specificity of prion transmission. The results of the in vivo experiments are compared in cells of different yeast species to determine whether species-specific patterns of intracellular environment influence the parameters of the prion species barrier. Further, in vitro aggregation experiments are performed to determine whether anions of the Hofmeister series influence cross-species specificity of prion transmission. Our results are consistent with the notion that protein sequence and conformation remain the primary determinants of cell specificity, whereas environmental conditions influence specificity primarily via favoring the formation of different prion strains.

      Experimental Procedures

      Yeast Strains and Plasmids

      Saccharomyces cerevisiae strains

      The S. cerevisiae strain GT17 (MATa ade1–14 his3 leu2 trp1 ura3 [psi pin]) was used for transfection of in vitro generated aggregates (
      • Chen B.
      • Newnam G.P.
      • Chernoff Y.O.
      Prion species barrier between the closely related yeast proteins is detected despite coaggregation.
      ,
      • Rubin J.
      • Khosravi H.
      • Bruce K.L.
      • Lydon M.E.
      • Behrens S.H.
      • Chernoff Y.O.
      • Bommarius A.S.
      Ion-specific effects on prion nucleation and strain formation.
      ). The strain GT256-23C (MATα ade1–14 his3Δ (or 11,15) lys2 leu2–3,112 trp1 ura3–52 sup35::HIS3 [PSI+ PIN+] [CEN LEU2 SUP35Sc]) was used for all shuffle experiments performed in the S. cerevisiae species. This strain harbors a strong prion variant. The S. cerevisiae strains GT797 (MATa ade1–14 his3Δ (or 11,15) lys2 ura3–52 leu2–3,112 trp1 sup35::HIS3 [CEN URA3 SUP35Sp] [psi pin]) or GT987 (MATa ade1–14 his3Δ (or 11,15) lys2 ura3–52 leu2–3,112 trp1 sup35::HIS3 [URA3 SUP35Sb] [psi pin]), bearing, respectively, the SUP35 genes from S. paradoxus or S. bayanus, were used as recipients for transfection with in vitro generated S. paradoxus or S. bayanus Sup35NM amyloids.

      Plasmids

      Centromeric (low-copy) vectors having URA3, LEU2, or LYS2 markers and bearing the divergent or chimeric SUP35 genes under the control of the endogenous S. cerevisiae SUP35 promoter (PSUP35) were employed in the plasmid shuffle experiments. Construction of the chimeric SUP35 derivatives has been described previously (
      • Chen B.
      • Newnam G.P.
      • Chernoff Y.O.
      Prion species barrier between the closely related yeast proteins is detected despite coaggregation.
      ,
      • Chen B.
      • Bruce K.L.
      • Newnam G.P.
      • Gyoneva S.
      • Romanyuk A.V.
      • Chernoff Y.O.
      Genetic and epigenetic control of the efficiency and fidelity of cross-species prion transmission.
      ). Plasmids pFA6a-kanMX6 (
      • Longtine M.S.
      • McKenzie III, A.
      • Demarini D.J.
      • Shah N.G.
      • Wach A.
      • Brachat A.
      • Philippsen P.
      • Pringle J.R.
      Additional modules for versatile and economical PCR-based gene deletion and modification in Saccharomyces cerevisiae.
      ), pBluescript-URA3 I (constructed by J. Kumar in the Chernoff laboratory), pRS303N (
      • Taxis C.
      • Knop M.
      System of centromeric, episomal, and integrative vectors based on drug resistance markers for Saccharomyces cerevisiae.
      ), and pRS303H (
      • Taxis C.
      • Knop M.
      System of centromeric, episomal, and integrative vectors based on drug resistance markers for Saccharomyces cerevisiae.
      ) were used as a source of marker genes to be introduced into S. paradoxus (see below). Plasmid pRS316 was used in the S. paradoxus ade1–14Sc construction (as described below) for co-transformation with the PCR product.

      S. paradoxus Strain Construction

      Natural strains of S. paradoxus are non-mating homothallic diploids lacking genetic markers that can be used for transformation and prion detection. To perform prion studies in S. paradoxus, we constructed genetically marked haploid heterothallic S. paradoxus strains bearing the ade1–14 (UGA) reporter (see above), which allows for [PSI+] detection. This construction included a multistep process, outlined below.

      Generation of lys2 and ura3-P2 Mutations

      The lys2 and ura3 auxotrophic mutations were introduced into the homothallic diploid S. paradoxus strain SP7-1D (provided by G. Naumov) by irradiation with UV light for 15–45 s and selecting mutants on α-aminoadipic- or 5-fluoroorotic acid-containing medium, respectively. Mutant alleles with the lowest reversion frequencies were chosen for further work. Diploid strains containing each allele were sporulated, and the resulting haploid spores were mated to generate double-heterozygous diploids. These diploids were sporulated and dissected, and spore clones unable to grow on both the medium lacking uracil (-Ura) and medium lacking lysine (-Lys) were identified. The respective mutant strains were sporulated, and spores were mated to obtain diploids heterozygous by both lys2 and ura3 alleles. These diploids were, in turn, sporulated to obtain homozygous double lys2 ura3 mutant strains.

      Generation of Heterothallic S. paradoxus Strains

      The initially homothallic lys2 ura3 S. paradoxus strain GT749-1B, generated as described previously, was used to produce heterothallic strains via replacement of the HO gene with the bacterial kanMX6 gene (Fig. 1A). For this purpose, the kanMX6 gene was PCR-amplified from plasmid pFA6a-kanMX6 (
      • Longtine M.S.
      • McKenzie III, A.
      • Demarini D.J.
      • Shah N.G.
      • Wach A.
      • Brachat A.
      • Philippsen P.
      • Pringle J.R.
      Additional modules for versatile and economical PCR-based gene deletion and modification in Saccharomyces cerevisiae.
      ), with primers having 50-bp extensions homologous to flanking regions of the S. paradoxus HO gene on both sides. This PCR fragment was used to transform the GT749-1B S. paradoxus strain. Replacement of HO by kanMX6 as a result of homologous recombination conveyed G418 resistance to the yeast cells and was verified by PCR. The resulting heterozygous HO/ho::KANMX6 strain was sporulated and dissected to produce heterothallic haploid ho::KANMX6 lys2 ura3 strains of both mating types.
      Figure thumbnail gr1
      FIGURE 1.The steps in construction of the S. paradoxus strains. A, the HO gene was disrupted by replacement with the bacterial KANMX6 gene. B, the ADE1 gene in S. paradoxus was disrupted by the URA3 gene from S. cerevisiae (URA3Sc). C, the ade1–14 and ura3 mutations were engineered by replacing URA3Sc (previously inserted to disrupt ADE1) with ade1–14Sc. D, the SUP35 gene in a S. paradoxus chromosome was replaced by natNT2 (conveying resistance to nourseothricin). E, an S. paradoxus strain having both ade1–14 and sup35Δ::natNT2 in its genotype was obtained by crossing two S. paradoxus yeast strains (one containing ade1–14, and another containing sup35Δ::natNT2 and a wild-type copy of SUP35 on a plasmid) of the opposite mating type. This was followed by sporulation and dissection.

      Generation of the ade1–14Sc Allele in S. paradoxus

      Next, the endogenous S. paradoxus wild-type ADE1 allele was replaced by the S. cerevisiae ade1–14 (UGA) mutant allele (ade1–14Sc). This enabled us to detect the presence of [PSI+] by read-through of ade1–14 (
      • Liebman S.W.
      • Chernoff Y.O.
      Prions in yeast.
      ) and compare the [PSI+] strains obtained in S. cerevisiae and S. paradoxus by using one and the same reporter. This construction was performed in two steps: replacement of endogenous S. paradoxus ADE1 by the S. cerevisiae URA3 gene (Fig. 1B) followed by replacement of URA3 by the S. cerevisiae ade1–14 allele (Fig. 1C). For the initial ADE1 replacement with URA3, the S. cerevisiae URA3 gene was PCR-amplified from the pBluescript-URA3 I plasmid (see above) using primers with 40-bp 5′ extensions homologous to the flanks of S. paradoxus ADE1 and transformed into a haploid S. paradoxus ho::KANMX6 lys2 ura3 strain constructed as described above. The transformants with ade1Δ::URA3 replacement were selected on -Ura medium and confirmed to become Ade. For the subsequent replacement of URA3 with ade1–14Sc, the ade1–14Sc allele was PCR-amplified from the S. cerevisiae genome using primers with 60-bp 5′ extensions homologous to the flanking regions of previously inserted URA3 in the S. paradoxus genome, followed by a second round of PCR aimed at further increasing the length of the homologous flanking regions by an additional 80 bp on each side for more efficient homologous recombination. The resulting PCR product containing 140 bp flanking extensions was co-transformed together with the LYS2 plasmid pRS317 (used to enrich the sample by transformants versus revertants) into a haploid ade1Δ::URA3 lys2 ura3 S. paradoxus strain constructed as described above. Transformants that were selected on -Lys medium with 5-fluoroorotic acid media and subsequently tested on -Ade and -Ura media. Ura- Ade- transformants were cured of the LYS2 plasmid and verified by PCR amplification and sequencing of the ade1–14Sc allele.

      Generation of the S. paradoxus Strains with SUP35 Genes of Different Origins

      For this purpose, the chromosomal SUP35 allele of the S. paradoxus strain was replaced by natNT2, conveying nourseothricin resistance, and PCR amplified from plasmid pRS303N (
      • Taxis C.
      • Knop M.
      System of centromeric, episomal, and integrative vectors based on drug resistance markers for Saccharomyces cerevisiae.
      ) by using primers with 40-bp 5′ extensions homologous to flanking regions of the S. paradoxus SUP35 gene (Fig. 1D). The PCR fragment was transformed into a diploid HO/ho::kanMX6 lys2 ura3 S. paradoxus strain homozygous for wild-type SUP35 and incorporated by homologous recombination in place of one of the alleles. The diploid also contained an additional copy of SUP35 on a centromeric plasmid. Transplacement was followed by sporulation and dissection to obtain haploid sup35Δ::natNT2 spores kept alive by the SUP35 plasmid. Such a spore clone was mated to the ade1–14Sc lys2 ura3 strain constructed as described earlier, followed by sporulation and dissection (Fig. 1E). The resulting haploid strain contained the only functional SUP35 copy on a plasmid. Therefore, derivatives with the SUP35 genes of various origins could be generated by introducing the plasmid with the respective SUP35 gene and subsequently losing the original SUP35 plasmid (this procedure is known as plasmid shuffle and is described in more detail below). As the final result of these manipulations, the S. paradoxus GT1320-36A strain containing the S. cerevisiae SUP35 gene was produced with the following genotype: MATa ade1Δ::ade1–14Sc (UGA) lys2 ura3-P2 Δsup35::natN2 ho::kanMX6 [SUP35Sc LYS2] [psi pin]. To generate a [PSI+] version of this strain, it was transfected with the cell extracts of S. cerevisiae strain GT256-23C as described below.

      Yeast Cultivation and Genetic Techniques

      Cultivation

      Standard yeast media and growth conditions were employed (
      • Kaiser C.
      • Michaelis S.
      • Mitchell A.
      ). Yeast cultures were incubated at 30 °C. Standard procedures (
      • Chernoff Y.O.
      • Uptain S.M.
      • Lindquist S.L.
      Analysis of prion factors in yeast.
      ) were used for [PSI+] detection, characterization, and curing by guanidine hydrochloride. All strains have an ade1–14 nonsense mutation in the ADE1 gene that contains a premature UGA stop codon, allowing detection of [psi] and [PSI+] through the read-through or nonsense suppression assay (
      • Chernoff Y.O.
      • Uptain S.M.
      • Lindquist S.L.
      Analysis of prion factors in yeast.
      ).

      Yeast Transformation and Transfection

      Standard techniques for yeast transformation were employed (
      • Kaiser C.
      • Michaelis S.
      • Mitchell A.
      ,
      • Chernoff Y.O.
      • Uptain S.M.
      • Lindquist S.L.
      Analysis of prion factors in yeast.
      ). For transfection with yeast extracts, cells of the [PSI+] donor strain were disrupted via a standard glass bead lysis procedure, and the resulting lysate was transfected into the spheroplasts of the [psi] S. paradoxus strain GT1320-36A using a modified protocol described by Rubin et al. (
      • Rubin J.
      • Khosravi H.
      • Bruce K.L.
      • Lydon M.E.
      • Behrens S.H.
      • Chernoff Y.O.
      • Bommarius A.S.
      Ion-specific effects on prion nucleation and strain formation.
      ) and originally adapted from Tanaka et al. (
      • Tanaka M.
      • Chien P.
      • Naber N.
      • Cooke R.
      • Weissman J.S.
      Conformational variations in an infectious protein determine prion strain differences.
      ). To prepare spheroplasts, the cell wall of a [psi] recipient cell was fragmented with zymolase. An empty URA3 vector was co-transfected into the recipient cells as an indicator that material had passed across the cell membrane (Fig. 2A). Transfectants were obtained on the medium lacking both uracil and tryptophane, and counterselecting against the Trp donor strain (-Ura-Trp) to avoid contamination by donor cells. Both small and large colonies were observed on -Ura-Trp. Only large colonies contained the URA3 plasmid, whereas smaller colonies (without the plasmid) resulted from a background growth, possibly because of a low concentration of YPD present in the transfectant selection medium. The large Ura+ colonies were tested on the medium lacking adenine (-Ade) to check for [PSI+] (Fig. 2B).
      Figure thumbnail gr2
      FIGURE 2.Cross-species prion transfection. A, summary of the transfection procedure. B, cellular extract was transfected from either a strong or weak [PSI+] S. cerevisiae strain into a [psi] S. paradoxus strain, expressing the Sup35 protein from S. cerevisiae. Representative [PSI+] S. paradoxus transfectants obtained from either the strong (left) or weak (right) [PSI+] donor strains are shown. Yeast cells were grown on YPD (for the color assay) and on -Ade medium (for the suppression assay) at 30 °C for 8 days.
      Transfection of in vitro generated aggregates into S. cerevisiae strains GT797 and GT987 was performed as described previously (
      • Rubin J.
      • Khosravi H.
      • Bruce K.L.
      • Lydon M.E.
      • Behrens S.H.
      • Chernoff Y.O.
      • Bommarius A.S.
      Ion-specific effects on prion nucleation and strain formation.
      ), with optional sonication to increase the frequency of transfection and using an empty plasmid with the LYS2 marker for selection of transfectants.

      Direct and Reverse Shuffle Procedures

      The SUP35 genes of various origins were exchanged in the S. paradoxus strain, constructed as described above, by using a plasmid shuffle procedure (Fig. 3) performed as described previously for S. cerevisiae (
      • Chen B.
      • Newnam G.P.
      • Chernoff Y.O.
      Prion species barrier between the closely related yeast proteins is detected despite coaggregation.
      ,
      • Chen B.
      • Bruce K.L.
      • Newnam G.P.
      • Gyoneva S.
      • Romanyuk A.V.
      • Chernoff Y.O.
      Genetic and epigenetic control of the efficiency and fidelity of cross-species prion transmission.
      ), except that LYS2 and URA3 markers were used on plasmids instead of LEU2 and URA3. For direct shuffle, cells containing a SUP35Sc LYS2 plasmid were transformed with a plasmid containing the SUP35 gene of the same or different origin and an URA3 marker, followed by loss of the original LYS2 plasmid. From each individual [PSI+] transformant, only a single Ura+ Lys colony was analyzed. To perform a reverse shuffle, cells obtained from the direct shuffle and containing the SUP35Sp URA3 plasmid were transformed with the SUP35Sc LYS2 plasmid and cured of the original URA3 plasmid.
      Figure thumbnail gr3
      FIGURE 3.Plasmid shuffle schematic. Shown is a schematic of direct and reverse plasmid shuffle. Sc, SUP35 from S. cerevisiae; PrDX, SUP35 genes of various origins or chimeric constructs.

      In Vitro Techniques

      Expression and Purification of Sup35NM

      Plasmid constructs containing the SUP35NM coding regions of different origins with the attached C-terminal His6 tags were generated as described previously (
      • Allen K.D.
      • Wegrzyn R.D.
      • Chernova T.A.
      • Müller S.
      • Newnam G.P.
      • Winslett P.A.
      • Wittich K.B.
      • Wilkinson K.D.
      • Chernoff Y.O.
      Hsp70 chaperones as modulators of prion life cycle: novel effects of Ssa and Ssb on the Saccharomyces cerevisiae prion [PSI+].
      ) and expressed in Escherichia coli host strain HMS174 (DE3) pLysS (Novagen) to produce the Sup35NM-(His6) proteins. The cell pellets were stored at −80 °C until purification. Purification of Sup35NM proteins from E. coli was carried out as described previously (
      • Yeh V.
      • Broering J.M.
      • Romanyuk A.
      • Chen B.
      • Chernoff Y.O.
      • Bommarius A.S.
      The Hofmeister effect on amyloid formation using yeast prion protein.
      ). The purified protein was precipitated using methanol at −20 °C and stored at −80 °C in 80% methanol.

      Kinetic Assays Using Thioflavin T

      The protein pellet stored at −80 °C was collected by centrifugation. The supernatant was discarded, and the protein was resuspended in 8 m urea. Sup35NM was then concentrated by a 10-kDa centrifugal filter and diluted 100-fold with PBS (pH 7.4). The samples were boiled for about 10 min before starting the aggregation experiments to break down any preformed aggregates. 1 mm thioflavin T (Sigma-Aldrich) solution was prepared fresh in PBS. Aggregation experiments were conducted in triplicates in a 96-well plate with final thioflavin T and Sup35NM concentrations of 100 and 10 μm, respectively, in the presence of 0.4 m sodium salt. The seeded experiments contained 5% by volume of sonicated preformed amyloid. Polymerization was carried out at 25 °C in a 96-well plate by shaking it linearly at 17 Hz in a Synergy H4 hybrid multimode microplate reader (BioTek, Winooski, VT). Fluorescence readings were taken every minute for about 13 h using an excitation wavelength of 440 nm and emission wavelength of 480 nm.

      Preparation of Amyloid Seeds

      The respective sodium salt was added to Sup35NM, purified, and prepared as described above to a final concentration of 0.5 m salt and 10 μm protein in a microcentrifuge tube. The samples were allowed to rotate at 20 rpm at room temperature for 2 days. After polymerization, the amyloid samples were stored at −80 °C until use.

      Discussion

      By constructing a unique set of S. paradoxus strains allowing for prion detection and using the transfection protocol, we were able to obtain S. cerevisiae and S. paradoxus cultures with one and the same strain of prion that helped us differentiate the effects of prion protein properties and intracellular environment on cross-species prion conversion. Our data show that both transmission barrier and conformational fidelity in vivo are primarily determined by the protein itself rather than by the environment. Therefore, differences between the S. cerevisiae and S. paradoxus intracellular environments do not affect major rules of [PSI+] transmission, although they might influence the quantitative characteristics of the process.
      The relative stability of the species barrier characteristics in the [PSI+] strain used in our studies differs somewhat from the results of Bateman and Wickner (
      • Bateman D.A.
      • Wickner R.B.
      The [PSI+] prion exists as a dynamic cloud of variants.
      ), who observed that parameters of [PSI+] transmission specificity could fluctuate reversibly during mitotic propagation of one and the same original prion strain even in the same S. cerevisiae intracellular environments. Authors interpreted these data as evidence for strain instability and existence of a “strain cloud.” However, other researchers (
      • Huang Y.-W.
      • Chang Y.-C.
      • Diaz-Avalos R.
      • King C.-Y.
      W8, a new Sup35 prion strain, transmits distinctive information with a conserved assembly scheme.
      ) did not see such fluctuations in the same experimental system for [PSI+] strains used in their studies, indicating that the existence of the readily detectable cloud is not a general rule. It should be noted that, in our experiments, we analyzed only one colony from each original transformant. Therefore, even if rare clonal variations in transmission specificity occurred, then they would have a minimal effect in our case. Notably, Bateman and Wickner (
      • Bateman D.A.
      • Wickner R.B.
      The [PSI+] prion exists as a dynamic cloud of variants.
      ) used the model of an “intraspecies” barrier controlled by variations within module III of Sup35 PrD. This module does not play a significant role in the cross-species transmission differences detected in our work. It is therefore possible that intraspecies transmission barriers are based on the different molecular foundations compared with the cross-species barriers studied in our work. Moreover, it is not known whether the differences between the components of a cloud (that is, differences between interconvertible “substrains”) are determined by the same structural mechanisms that control differences between prion strains that are firmly established and faithfully reproduced at the phenotypic level.
      Because S. cerevisiae and S. paradoxus species are relatively closely related to each other, it would be of interest to determine whether more drastic differences in the intracellular environment could influence the specificity of prion transmission. We engineered the system for [PSI+] detection in S. bayanus and showed that the [PSI+] prion can be transfected from the S. cerevisiae to S. bayanus strains.
      K. L. Bruce, B. Chen, S. Gyoneva, and Y. O. Chernoff, unpublished data.
      However, the systematic analysis of cross-species conversion in S. bayanus cells turned out to be impossible because of the overall low efficiency of the nonsense suppression assay in this species (data not shown). While continuing the optimization of the S. bayanus-based prion detection system, we moved the cross-species prion transmission experiments to an entirely different environment by performing in vitro experiments in solutions of various ionic compositions.
      We have reported previously that anions of the Hofmeister series influence the kinetics and strain preference during in vitro amyloid formation by Sup35NMSc (
      • Rubin J.
      • Khosravi H.
      • Bruce K.L.
      • Lydon M.E.
      • Behrens S.H.
      • Chernoff Y.O.
      • Bommarius A.S.
      Ion-specific effects on prion nucleation and strain formation.
      ,
      • Yeh V.
      • Broering J.M.
      • Romanyuk A.
      • Chen B.
      • Chernoff Y.O.
      • Bommarius A.S.
      The Hofmeister effect on amyloid formation using yeast prion protein.
      ), and now we show that Sup35NM proteins from the other species of the Saccharomyces sensu stricto group, Sup35NMSb and Sup35NMSp, also respond to the presence of kosmotropic or chaotropic ions in the same way as Sup35NMSc. We confirmed previous data by showing that, in vitro, in agreement with previous research, the more similar Sc/Sp proteins exhibit a relatively low barrier in the transmission of the prion state, whereas the more divergent Sb protein shows a high barrier with the other two proteins (
      • Chen B.
      • Newnam G.P.
      • Chernoff Y.O.
      Prion species barrier between the closely related yeast proteins is detected despite coaggregation.
      ). We also performed systematic studies of the effects of the Hofmeister series anions on cross-species prion transmission. Overall, our data show that the salt present during amyloid formation can alter the parameters of the species barrier. The simplest explanation for such a result is that salts present during seed formation determine the seed conformation, which, in turn, influences the efficiency of the conformational adaptation of the added monomer to the preformed nuclei provided by this seed. This agrees with our previous in vivo data showing that the prion strain pattern controls the specificity of transmission of the prion state to the newly immobilized protein (
      • Chen B.
      • Bruce K.L.
      • Newnam G.P.
      • Gyoneva S.
      • Romanyuk A.V.
      • Chernoff Y.O.
      Genetic and epigenetic control of the efficiency and fidelity of cross-species prion transmission.
      ). In contrast, salts present during the process of cross-seeding exhibit a strong and systematic influence on the kinetics of cross-species aggregation in accordance with the inverse Hofmeister trend that has been reported previously for homologous aggregation (
      • Rubin J.
      • Khosravi H.
      • Bruce K.L.
      • Lydon M.E.
      • Behrens S.H.
      • Chernoff Y.O.
      • Bommarius A.S.
      Ion-specific effects on prion nucleation and strain formation.
      ), but they do not alter the transmission specificity.
      Interestingly, we observed that seeds formed in chloride are more efficient in promoting both homologous and cross-species aggregation than seeds formed in sulfate or perchlorate (Figs. 8C and 10). We hypothesize that this might be because the seed nucleation in chloride produces a most diverse mix of various prion strains. This diversity helps to provide a pool of seed conformations, some of which are more amenable for the monomer to template onto. Therefore, it is always possible to select a fraction of strains that can act as highly efficient templates under any given conditions. By contrast, aggregation in the presence of either highly kosmotropic (e.g. sulfate) or highly chaotropic (e.g. perchlorate) salt exhibits a more pronounced bias toward one particular strain. Therefore, the type of the strain “preadapted” to the changed environment might be lacking.
      Previous work by the Weissman group (
      • Tanaka M.
      • Chien P.
      • Naber N.
      • Cooke R.
      • Weissman J.S.
      Conformational variations in an infectious protein determine prion strain differences.
      ,
      • Tanaka M.
      • Chien P.
      • Yonekura K.
      • Weissman J.S.
      Mechanism of cross-species prion transmission: an infectious conformation compatible with two highly divergent yeast prion proteins.
      ,
      • Chien P.
      • DePace A.H.
      • Collins S.R.
      • Weissman J.S.
      Generation of prion transmission barriers by mutational control of amyloid conformations.
      ) has shown that temperature can be used to affect the specificity of the species barrier between S. cerevisiae and Candida albicans. Tanaka et al. (
      • Tanaka M.
      • Chien P.
      • Naber N.
      • Cooke R.
      • Weissman J.S.
      Conformational variations in an infectious protein determine prion strain differences.
      ) have observed that, when Sup35NMSc is aggregated at low (4 °C) temperature, more “promiscuous” aggregates with a stronger phenotype and higher seeding capabilities can be generated compared with aggregates produced at a high (37 °C) temperature. Tanaka et al. (
      • Tanaka M.
      • Chien P.
      • Yonekura K.
      • Weissman J.S.
      Mechanism of cross-species prion transmission: an infectious conformation compatible with two highly divergent yeast prion proteins.
      ) have shown that the seeds of the S. cerevisiae Sup35NM protein formed at different temperatures show a variable ability to cross-seed the Sup35NM protein from C. albicans. Chien et al. (
      • Chien P.
      • DePace A.H.
      • Collins S.R.
      • Weissman J.S.
      Generation of prion transmission barriers by mutational control of amyloid conformations.
      ) have shown that polymerization of a chimeric protein, combining the regions from the Sup35 prion domains of both S. cerevisiae and C. albicans, can produce distinct prion strains with different seeding specificities depending on temperature. These results agree with our observations that the differences in ionic composition influence the species barrier by promoting the formation of different strains. However, our work represents the first systematic comparison of the effects of aggregation conditions at the stages of initial aggregate formation and cross-species seeding. Our data confirm that protein sequence and conformation play a central role in determining the specificity of prion transmission and show that external factors influence transmission specificity primarily by altering the nature of the initial seed, whereas the conditions of the actual cross-seeding reaction itself have an effect only on the kinetics of the process.

      Author Contributions

      A. S. B. and Y. O. C. designed the study. A. S. and K. L. B. performed the experiments. B. C., S. G., and K. L. B. constructed the strains. A. S., K. L. B., S. H. B., A. S. B., and Y. O. C. analyzed the results and wrote and edited the manuscript.

      Acknowledgments

      We thank D. Deng, K. Jang, and A. Tippur for help with some experiments and A. Grizel, G. Newnam, and A. Romanyuk for helpful discussions.

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