title: The prion domain of yeast Ure2p induces autocatalytic formation of amyloid fibers by a recombinant fusion protein.
abstract: The Ure2 protein from Saccharomyces cerevisiae has been proposed to undergo a prion-like autocatalytic conformational change, which leads to inactivation of the protein, thereby generating the [URE3] phenotype. The first 65 amino acids, which are dispensable for the cellular function of Ure2p in nitrogen metabolism, are necessary and sufficient for [URE3] (Masison & Wickner, 1995), leading to designation of this domain as the Ure2 prion domain (UPD). We expressed both UPD and Ure2 as glutathione-S-transferase (GST) fusion proteins in Escherichia coli and observed both to be initially soluble. Upon cleavage of GST-UPD by thrombin, the released UPD formed ordered fibrils that displayed amyloid-like characteristics, such as Congo red dye binding and green-gold birefringence. The fibrils exhibited high beta-sheet content by Fourier transform infrared spectroscopy. Fiber formation proceeded in an autocatalytic manner. In contrast, the released, full-length Ure2p formed mostly amorphous aggregates; a small amount polymerized into fibrils of uniform size and morphology. Aggregation of Ure2p could be seeded by UPD fibrils. Our results provide biochemical support for the proposal that the [URE3] state is caused by a self-propagating inactive form of Ure2p. We also found that the uncleaved GST-UPD fusion protein could polymerize into amyloid fibrils by a strictly autocatalytic mechanism, forcing the GST moiety of the protein to adopt a new, beta-sheet-rich conformation. The findings on the GST-UPD fusion protein indicate that the ability of the prion domain to mediate a prion-like conversion process is not specific for or limited to the Ure2p.
authors.affiliation: Institute for Neurodegenerative Diseases, University of California, San Francisco 94143-0518, USA.
authors.forename: M
authors.initials: M
authors.lastname: Schlumpberger
authors.forename: H
authors.initials: H
authors.lastname: Wille
authors.forename: M A
authors.initials: MA
authors.lastname: Baldwin
authors.forename: D A
authors.initials: DA
authors.lastname: Butler
authors.forename: I
authors.initials: I
authors.lastname: Herskowitz
authors.forename: S B
authors.initials: SB
authors.lastname: Prusiner
mesh_terms: D000682:Amyloid / Q000737:chemistry* / Q000648:ultrastructure; D004396:Coloring Agents; D003224:Congo Red; D005656:Fungal Proteins / Q000737:chemistry* / Q000235:genetics; D005979:Glutathione Peroxidase; D005982:Glutathione Transferase / Q000235:genetics; D008854:Microscopy, Electron; D011328:Prions / Q000737:chemistry* / Q000235:genetics; D017433:Protein Structure, Secondary; D011993:Recombinant Fusion Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000648:ultrastructure; D012441:Saccharomyces cerevisiae / Q000737:chemistry; D029701:Saccharomyces cerevisiae Proteins; D017550:Spectroscopy, Fourier Transform Infrared
chemical_list: D000682:Amyloid; D004396:Coloring Agents; D005656:Fungal Proteins; D011328:Prions; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; D003224:Congo Red; D005979:Glutathione Peroxidase; C067020:URE2 protein, S cerevisiae; D005982:Glutathione Transferase