NR APEZ
AU Baxa,U.; Taylor,K.L.; Wall,J.S.; Simon,M.N.; Cheng,N.; Wickner,R.B.; Steven,A.C.
TI Architecture of Ure2p prion filaments. The N-terminal domains form a central core fiber.
QU The Journal of Biological Chemistry 2003 Oct 31; 278(44): 43717-27
IA http://www.jbc.org/cgi/content/full/278/44/43717
PT journal article
AB The [URE3] prion is an inactive, self-propagating, filamentous form of the Ure2 protein, a regulator of nitrogen catabolism in yeast. The N-terminal "prion" domain of Ure2p determines its in vivo prion properties and in vitro amyloid-forming ability. Here we determined the overall structures of Ure2p filaments and related polymers of the prion domain fused to other globular proteins. Protease digestion of 25-nm diameter Ure2p filaments trimmed them to 4-nm filaments, which mass spectrometry showed to be composed of prion domain fragments, primarily residues approximately 1-70. Fusion protein filaments with diameters of 14-25 nm were also reduced to 4-nm filaments by proteolysis. The prion domain transforms from the most to the least protease-sensitive part upon filament formation in each case, implying that it undergoes a conformational change. Intact filaments imaged by cryo-electron microscopy or after vanadate staining by scanning transmission electron microscopy (STEM) revealed a central 4-nm core with attached globular appendages. STEM mass per unit length measurements of unstained filaments yielded 1 monomer per 0.45 nm in each case. These observations strongly support a unifying model whereby subunits in Ure2p filaments, as well as in fusion protein filaments, are connected by interactions between their prion domains, which form a 4-nm amyloid filament backbone, surrounded by the corresponding C-terminal moieties.
MH Blotting, Western; Chromatography, Liquid; Cloning, Molecular; Cryoelectron Microscopy; Electrophoresis, Polyacrylamide Gel; Endopeptidase K/metabolism; Microscopy, Electron; Microscopy, Electron, Scanning Transmission; Polymers/chemistry; Prions/*chemistry; Protein Conformation; Protein Structure, Tertiary; Saccharomyces cerevisiae Proteins/*chemistry; Spectrum Analysis, Mass; Time Factors; Trypsin/chemistry
AD Laboratories of Structural Biology, National Institute of Arthritis, Musculoskeletal, and Skin Diseases, and Biochemistry and Genetics, NIDDK, National Institutes of Health, Bethesda, Maryland 20892, USA
SP englisch
PO USA