Architecture of Ure2p prion filaments: the N-terminal domains form a central core fiber.
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.
Baxa
Ulrich
U
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.
Taylor
Kimberly L
KL
Wall
Joseph S
JS
Simon
Martha N
MN
Cheng
Naiqian
N
Wickner
Reed B
RB
Steven
Alasdair C
AC
D015153:Blotting, Western; D002853:Chromatography, Liquid; D003001:Cloning, Molecular; D020285:Cryoelectron Microscopy; D004591:Electrophoresis, Polyacrylamide Gel; D019286:Endopeptidase K / Q000378:metabolism; D005979:Glutathione Peroxidase; D013058:Mass Spectrometry; D008854:Microscopy, Electron; D017348:Microscopy, Electron, Scanning Transmission; D011108:Polymers / Q000737:chemistry; D011328:Prions / Q000737:chemistry*; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry*; D013997:Time Factors; D014357:Trypsin / Q000737:chemistry
D011108:Polymers; D011328:Prions; D029701:Saccharomyces cerevisiae Proteins; D005979:Glutathione Peroxidase; C067020:URE2 protein, S cerevisiae; D014357:Trypsin; D019286:Endopeptidase K