NR AKNI
AU Schlumpberger,M.; Wille,H.; Baldwin,M.A.; Butler,D.A.; Herskowitz,I.; Prusiner,S.B.
TI The prion domain of yeast Ure2p induces autocatalytic formation of amyloid fibers by a recombinant fusion protein
QU Protein Science 2000 Mar; 9(3): 440-51
IA http://www.proteinscience.org/cgi/reprint/9/3/440
PT journal article
AB 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.
MH Amyloid/*chemistry/ultrastructure; Congo Red; Dyes; Fungal Proteins/*chemistry/genetics; Glutathione Transferase/genetics; Microscopy, Electron; Prions/*chemistry/genetics; Protein Structure, Secondary; Recombinant Fusion Proteins/*chemistry/genetics/isolation &; purification/ultrastructure; Saccharomyces cerevisiae/chemistry; Spectroscopy, Fourier Transform Infrared; Support, Non-U.S. Gov't
AD Institute for Neurodegenerative Diseases, University of California, San Francisco 94143-0518, USA
SP englisch
PO USA