Prion generation in vitro: Amyloid of Ure2p is infectious

Laboratory of Biochemistry and Genetics, National Institute of Diabetes and Digestive and Kidney Diseases, Bethesda, MD 20892-0830, USA.
The EMBO Journal (Impact Factor: 10.43). 10/2005; 24(17):3082-92. DOI: 10.1038/sj.emboj.7600772
Source: PubMed


[URE3] is a prion (infectious protein) of the Ure2 protein of yeast. In vitro, Ure2p can form amyloid filaments, but direct evidence that these filaments constitute the infectious form is still missing. Here we demonstrate that recombinant Ure2p converted into amyloid can infect yeast cells lacking the prion. Infection produced a variety of [URE3] variants. Extracts of [URE3] strains, as well as amyloid of Ure2p formed in an extract-primed reaction could transmit to uninfected cells the [URE3] variant present in the cells from which the extracts were made. Infectivity and determinant of [URE3] variants resided within the N-terminal 65 amino acids of Ure2p. Notably, we could show that amyloid filaments of recombinant Ure2p are nearly as infectious per mass of Ure2p as extracts of [URE3] strains. Sizing experiments indicated that infectious particles in vitro and in vivo were >20 nm in diameter, suggesting that they were amyloid filaments and not smaller oligomeric structures. Our data indicate that there is no substantial difference between filaments formed in vivo and in vitro.

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Available from: Andreas Brachmann, Aug 14, 2014
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    • "By binding to transcription factor Gln3, Ure2 suppresses expression of genes required for uptake of poor nitrogen sources when a good nitrogen source is present. Amyloids formed from purified Ure2 and Sup35 prion domains possess in-register parallel beta sheet architecture (Tanaka et al. 2004; Brachmann et al. 2005; Shewmaker et al. 2006; Baxa et al. 2007; Ngo et al. 2011). "
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    ABSTRACT: The BAG family of proteins are evolutionarily conserved from yeast to humans and plants. In animals and plants, the BAG family possesses multiple members with overlapping and distinct functions that regulate many cellular processes such as signalling, protein degradation and stress response. The only BAG domain protein in Saccharomyces cerevisiae is Snl1, which is anchored to the endoplasmic reticulum through an amino-terminal transmembrane region. Snl1 is the only known membrane associated nucleotide exchange factor for Hsp70 and thus its role in regulating cytosolic Hsp70 functions is not clear. Here, we examine whether Snl1 regulates Hsp70 activity in the propagation of stable prion like protein aggregates. We show that unlike other nucleotide exchange factors, Snl1 is not required for propagation of yeast prions [URE3] and [PSI(+)]. Overexpressing Snl1 derivative consisting of only the BAG domain (Snl1-S) cures [URE3], however elevated level of entire cytosolic domain of Snl1 (Snl1-M), which has nine additional amino-terminal residues, has no effect. Substituting the three lysine residues in this region of Snl1-M with alanine restores ability to cure [URE3]. [PSI(+)] is unaffected by overproduction of either Snl1-S or Snl1-M. The Snl1-S mutant engineered with weaker affinity to Hsp70 does not cure [URE3], indicating that curing of [URE3] by Snl1-S requires Hsp70. Our data suggest that Snl1 anchoring to ER or nuclear membrane restricts its ability to modulate cytosolic activities of Hsp70 proteins. Furthermore, the short amino-terminal extension of the BAG domain profoundly affects its function.
    G3-Genes Genomes Genetics 01/2014; 4(3). DOI:10.1534/g3.113.009993 · 3.20 Impact Factor
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    • "[URE3], the longest-studied prion in S. cerevisiae, is the prion form of the nitrogen catabolism regulator Ure2. Like [PSI + ], a colorimetric [URE3] reporter system has been established (Brachmann et al. 2005). In this system, the ADE2 gene, another adenine marker, is placed under the chromosomal DAL5 promoter. "
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    ABSTRACT: Prions are epigenetic modifiers that cause partially loss-of-function phenotypes of the proteins in Saccharomyces cerevisiae. The molecular chaperone network that supports prion propagation in the cell has seen a great progress in the last decade. However, the cellular machinery to activate or deactivate the prion states remains an enigma, largely due to insufficient knowledge of prion-regulating factors. Here, we report that overexpression of a [PSI(+) ]-inducible Q/N-rich protein, Lsm4, eliminates the three major prions [PSI(+) ], [URE3], and [RNQ(+) ]. Subcloning analysis revealed that the Q/N-rich region of Lsm4 is responsible for the prion loss. Lsm4 formed an amyloid in vivo, which seemed to play a crucial role in the prion elimination. Fluorescence correlation spectroscopy analysis revealed that in the course of the Lsm4-driven [PSI(+) ] elimination, the [PSI(+) ] aggregates undergo a size increase, which ultimately results in the formation of conspicuous foci in otherwise [psi(-) ]-like mother cells. We also found that the antiprion activity is a general property of [PSI(+) ]-inducible factors. These data provoked a novel "unified" model that explains both prion induction and elimination by a single scheme.
    MicrobiologyOpen 06/2013; 2(3). DOI:10.1002/mbo3.83 · 2.21 Impact Factor
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    • "Saccharomyces cerevisiae strains used in this study are as follows: NPK50 ([PSI + ] [pin ) ] MATa ade1-14 leu2 ura3 his3 trp1), NPK200 ([psi ) ] [PIN + ] isogenic with NPK50), NPK294 ([PSI + ] [PIN + ] isogenic with NPK50), NPK299 (rnq1::URA3 derivative of NK294), NPK300 ([PIN + ] derivative of NPK50), NPK301 ([ure-o] [pin ) ] MATa PD-ADE2 his3 leu2 trp1 kar1 PD-CAN1), NPK302 ([URE3] [pin ) ] isogenic with NPK301), NPK435 ([PIN + ] derivative of NPK302), NPK372 ([psi ) ] [pin ) ] hsp104::LEU2 derivative of NPK200) (Brachmann et al. 2005; Kurahashi et al. 2008), NS42 ([PSI + ] [PIN + ] MATa ade1-14 leu2 ura3 his3 trp1 rnq1::KanMX [pRS416RNQ1p- RNQ1 (ARS ⁄ CEN, URA3 marker)]) (Shibata et al. 2009), NPK620 ([PSI + ] [PIN + ] MATa ade1-14 leu2 ura3 his3 trp1 sup35::SUP35-GFP) and NPK635 ([psi ) ] [PIN + ] isogenic with NPK620) (NPK620 and NPK635 are derivative of G74-D694 [psi ) ]), (Kawai-Noma et al. 2009). "
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    ABSTRACT: [PIN(+)] is the prion form of the Rnq1 protein of unknown function in Saccharomyces cerevisiae. A glutamine/asparagine (Q/N)-rich C-terminal domain is necessary for the propagation of [PIN(+)], whereas the N-terminal region is non-Q/N-rich and considered the nonprion domain. Here, we isolated numerous single-amino-acid mutations in Rnq1, phenotypically similar to Rnq1Δ100, which inhibit [PSI(+)] propagation in the [PIN(+)] state, but not in the [pin(-)] state, when overproduced. The dynamics of the prion aggregates was analyzed by semi-denaturing detergent-agarose gel electrophoresis and fluorescence correlation spectroscopy. The results indicated that [PSI(+)] aggregates were enlarged in mother cells and, instead, not apparently transmitted into daughter cells. Under these conditions, the activity of Hsp104, a known prion disaggregase, was not affected when monitored for the thermotolerance of the rnq1 mutants. These [PSI(+)]-inhibitory rnq1 mutations did not affect [PIN(+)] propagation itself when over-expressed from a strong promoter, but instead destabilized [PIN(+)] when expressed from the weak authentic RNQ1 promoter. The majority of these mutated residues are mapped to the surface, and on one side, of contiguous α-helices of the nonprion domain of Rnq1, suggesting its involvement in interactions with a prion or a factor necessary for prion development.
    Genes to Cells 04/2011; 16(5):576-89. DOI:10.1111/j.1365-2443.2011.01511.x · 2.81 Impact Factor
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