Prion generation in vitro: amyloid of Ure2p is infectious.
ABSTRACT [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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ABSTRACT: Proteins in the cell experience various stressful conditions that can affect their ability to attain and maintain the structural conformations they need to perform effectively. Protein chaperones are an important part of a cellular protein quality control system that protects the integrity of the proteome in the face of such challenges. Chaperones from different conserved families have multiple members that cooperate to regulate each other's activity and produce machines that perform a variety of tasks. The large numbers of related haperones with both functionally overlapping and distinct activities allows fine-tuning of the machinery for specific tasks, but presents a daunting degree of complexity. Yeast prions are misfolded forms of cellular proteins whose propagation depends on the action of protein chaperones. Studying how propagation of yeast prions is affected by alterations in functions of various chaperones provides an approach to understanding this complexity.Current Pharmaceutical Biotechnology 11/2014; 15(11). DOI:10.2174/1389201015666141103021035 · 2.51 Impact Factor
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ABSTRACT: Author Summary The cellular chaperone machinery helps proteins adopt and maintain native conformations and protects cells from stress. The yeast Hsp40s Ydj1 and Sis1 are co-chaperones that regulate Hsp70s, which are key components of many chaperone complexes. Both of these Hsp40s are crucial for growth and Ydj1 directs disaggregation activity of the Hsp100-based machinery to provide stress protection while Sis1 directs this activity to promote prion replication. Ydj1 also cures yeast of certain prions when overexpressed. We show that C-terminal domains that possess substrate-binding function of Ydj1 and Sis1 can mediate these and other functional distinctions and that the degree that prions depend on Sis1 activities could underlie differences in how they respond to alterations of chaperones. These findings support a view that Hsp40s regulate and specify functions of the chaperone machinery through substrate discrimination and cooperation with Hsp70. The disproportionate evolutionary expansion of Hsp40s (J-proteins) relative to their Hsp70 partners led to a proposal that this amplification allows increased regulation and fine-tuning of chaperone machines for increasingly complex processes. Our findings support this idea and provide insight into fundamental aspects of this cooperation.PLoS Genetics 10/2014; 10(10):e1004720. DOI:10.1371/journal.pgen.1004720 · 8.17 Impact Factor
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ABSTRACT: A prion is an infectious protein horizontally transmitting a disease or trait without a required nucleic acid. Yeast and fungal prions are nonchromosomal genes composed of protein, generally an altered form of a protein that catalyzes the same alteration of the protein. Yeast prions are thus transmitted both vertically (as genes composed of protein) and horizontally (as infectious proteins, or prions). Formation of amyloids (linear ordered β-sheet-rich protein aggregates with β-strands perpendicular to the long axis of the filament) underlies most yeast and fungal prions, and a single prion protein can have any of several distinct self-propagating amyloid forms with different biological properties (prion variants). Here we review the mechanism of faithful templating of protein conformation, the biological roles of these prions, and their interactions with cellular chaperones, the Btn2 and Cur1 aggregate-handling systems, and other cellular factors governing prion generation and propagation. Human amyloidoses include the PrP-based prion conditions and many other, more common amyloid-based diseases, several of which show prion-like features. Yeast prions increasingly are serving as models for the understanding and treatment of many mammalian amyloidoses. Patients with different clinical pictures of the same amyloidosis may be the equivalent of yeasts with different prion variants. Copyright © 2015, American Society for Microbiology. All Rights Reserved.Microbiology and molecular biology reviews: MMBR 03/2015; 79(1):1-17. DOI:10.1128/MMBR.00041-14 · 15.26 Impact Factor