title: Characterization of oligomeric species in the fibrillization pathway of the yeast prion Ure2p.
abstract: The [URE3] prion of Saccharomyces cerevisiae shares many features with mammalian prions and poly-glutamine related disorders and has become a model for studying amyloid diseases. The development of the [URE3] phenotype is thought to be caused by a structural switch in the Ure2p protein. In [URE3] cells, Ure2p is found predominantly in an aggregated state, while it is a soluble dimer in wild-type cells. In vitro, Ure2p forms fibrils with amyloid-like properties. Several studies suggest that the N-terminal domain of Ure2p is essential for prion formation. In this work, we investigated the fibril formation of Ure2p by isolating soluble oligomeric species, which are generated during fibrillization, and characterized them with respect to size and structure. Our data support the critical role of the N-terminal domain for fibril formation, as we observed fibrils in the presence of 5 M guanidinium chloride, conditions at which the C-terminal domain is completely unfolded. Based on fluorescence measurements, we conclude that the structure of the C-terminal domain is very similar in dimeric and fibrillar Ure2p. When studying the time course of fibrillization, we detected the formation of small, soluble oligomeric species during the early stages of the process. Their remarkable resistance against denaturants, their increased content of beta-structure, and their ability to 'seed' Ure2p fibrillization suggest that conversion to the amyloid-like conformation has already occurred. Thus, they likely represent critical intermediates in the fibrillization pathway of Ure2p.
authors.affiliation: Fachbereich Chemie, Technische Universität München, Lichtenbergstr. 4, D-85747 Garching, Germany.
authors.forename: Silvia
authors.initials: S
authors.lastname: Catharino
authors.forename: Johannes
authors.initials: J
authors.lastname: Buchner
authors.forename: Stefan
authors.initials: S
authors.lastname: Walter
mesh_terms: D002850:Chromatography, Gel; D002942:Circular Dichroism; D005979:Glutathione Peroxidase; D018625:Microscopy, Atomic Force; D011328:Prions / Q000737:chemistry / Q000378:metabolism*; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000378:metabolism*; D012995:Solubility; D013050:Spectrometry, Fluorescence
chemical_list: D011328:Prions; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; D005979:Glutathione Peroxidase; C067020:URE2 protein, S cerevisiae