Amyloid Nucleation and Hierarchical Assembly of Ure2p Fibrils

Abstract

The yeast prion protein Ure2 forms amyloid-like filaments in vivo and in vitro. This ability depends on the N-terminal prion domain, which contains Asn/Gln repeats, a motif thought to cause human disease by forming stable protein aggregates. The Asn/Gln region of the Ure2p prion domain extends to residue 89, but residues 15–42 represent an island of “normal” random sequence, which is highly conserved in related species and is relatively hydrophobic. We compare the time course of structural changes monitored by thioflavin T (ThT) binding fluorescence and atomic force microscopy for Ure2 and a series of prion domain mutants under a range of conditions. Atomic force microscopy height images at successive time points during a single growth experiment showed the sequential appearance of at least four fibril types that could be readily differentiated by height (5, 8, 12, or 9 nm), morphology (twisted or smooth), and/or time of appearance (early or late in the plateau phase of ThT binding). The Ure2 dimer (h = 2.6 ± 0.5 nm) and granular particles corresponding to higher order oligomers (h = 4–12 nm) could also be detected. The mutants 15Ure2 and Δ15–42Ure2 showed the same time-dependent variation in fibril types but with an increased lag time detected by ThT binding compared with wild-type Ure2. In addition, Δ15–42Ure2 showed reduced binding to ThT. The results imply a role of the conserved region in both amyloid nucleation and formation of the binding surface recognized by ThT. Further, Ure2 amyloid formation is a multistep process via a series of fibrillar intermediates. The yeast prion protein Ure2 forms amyloid-like filaments in vivo and in vitro. This ability depends on the N-terminal prion domain, which contains Asn/Gln repeats, a motif thought to cause human disease by forming stable protein aggregates. The Asn/Gln region of the Ure2p prion domain extends to residue 89, but residues 15–42 represent an island of “normal” random sequence, which is highly conserved in related species and is relatively hydrophobic. We compare the time course of structural changes monitored by thioflavin T (ThT) binding fluorescence and atomic force microscopy for Ure2 and a series of prion domain mutants under a range of conditions. Atomic force microscopy height images at successive time points during a single growth experiment showed the sequential appearance of at least four fibril types that could be readily differentiated by height (5, 8, 12, or 9 nm), morphology (twisted or smooth), and/or time of appearance (early or late in the plateau phase of ThT binding). The Ure2 dimer (h = 2.6 ± 0.5 nm) and granular particles corresponding to higher order oligomers (h = 4–12 nm) could also be detected. The mutants 15Ure2 and Δ15–42Ure2 showed the same time-dependent variation in fibril types but with an increased lag time detected by ThT binding compared with wild-type Ure2. In addition, Δ15–42Ure2 showed reduced binding to ThT. The results imply a role of the conserved region in both amyloid nucleation and formation of the binding surface recognized by ThT. Further, Ure2 amyloid formation is a multistep process via a series of fibrillar intermediates. Ure2p is the protein determinant of the epigenetic factor [URE3] of Saccharomyces cerevisiae, which has been demonstrated to represent a prion of yeast (1Wickner R.B. Science. 1994; 264: 566-569Google Scholar, 2Masison D.C. Wickner R.B. Science. 1995; 270: 93-95Google Scholar). Analogous to the mammalian prion protein (3Prusiner S.B. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 13363-13383Google Scholar), Ure2p is aggregated, inactive, and protease-resistant in prion strains (1Wickner R.B. Science. 1994; 264: 566-569Google Scholar, 2Masison D.C. Wickner R.B. Science. 1995; 270: 93-95Google Scholar). In addition, amyloid-like filaments are observed both in vivo (4Speransky V.V. Taylor K.L. Edskes H.K. Wickner R.B. Steven A.C. J. Cell Biol. 2001; 153: 1327-1335Google Scholar) and in vitro (5Taylor K.L. Cheng N. Williams R.W. Steven A.C. Wickner R.B. Science. 1999; 283: 1339-1342Google Scholar, 6Thual C. Komar A.A. Bousset L. Fernandez-Bellot E. Cullin C. Melki R. J. Biol. Chem. 1999; 274: 13666-13674Google Scholar). Fibrils formed in vitro show characteristic green birefringence on Congo red binding (5Taylor K.L. Cheng N. Williams R.W. Steven A.C. Wickner R.B. Science. 1999; 283: 1339-1342Google Scholar, 7Schlumpberger M. Wille H. Baldwin M.A. Butler D.A. Herskowitz I. Prusiner S.B. Protein Sci. 2000; 9: 440-451Google Scholar, 8Bousset L. Thomson N.H. Radford S.E. Melki R. EMBO J. 2002; 21: 2903-2911Google Scholar) and also bind thioflavin T (ThT) 1The abbreviations used are: ThTthioflavin TAFMatomic force spectroscopyEMelectron microscopyPrDprion domainWTwild-type. (7Schlumpberger M. Wille H. Baldwin M.A. Butler D.A. Herskowitz I. Prusiner S.B. Protein Sci. 2000; 9: 440-451Google Scholar, 8Bousset L. Thomson N.H. Radford S.E. Melki R. EMBO J. 2002; 21: 2903-2911Google Scholar, 9Zhu L. Zhang X.J. Wang L.Y. Zhou J.M. Perrett S. J. Mol. Biol. 2003; 328: 235-254Google Scholar), a dye considered highly specific for amyloid (10Levine H. Protein Sci. 1993; 2: 404-410Google Scholar, 11Khurana R. Uversky V.N. Nielsen L. Fink A.L. J. Biol. Chem. 2001; 276: 22715-22721Google Scholar). Amyloids are thought to form by conversion of the native protein structure to a generic cross-β structure, identified by a characteristic x-ray diffraction pattern (12Sunde M. Serpell L.C. Bartlam M. Fraser P.E. Pepys M.B. Blake C.C. J. Mol. Biol. 1997; 273: 729-739Google Scholar). However, an unusual property of Ure2 fibrils is their ability to maintain native-like ligand binding properties within the fibrillar arrays (8Bousset L. Thomson N.H. Radford S.E. Melki R. EMBO J. 2002; 21: 2903-2911Google Scholar, 13Baxa U. Speransky V. Steven A.C. Wickner R.B. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 5253-5260Google Scholar). Consistent with this, Ure2 fibrils formed by standing at subambient temperatures show a Fourier transform infrared spectrum consistent with native-like helical content (8Bousset L. Thomson N.H. Radford S.E. Melki R. EMBO J. 2002; 21: 2903-2911Google Scholar), and the cross-β x-ray diffraction band becomes apparent only after incubation of the protein close to the L. J. Melki R. J. Biol. 2003; Scholar). Atomic force microscopy of fibrils to be with a height of and a of (8Bousset L. Thomson N.H. Radford S.E. Melki R. EMBO J. 2002; 21: 2903-2911Google Scholar). thioflavin T atomic force microscopy prion domain Ure2 prion formation in vivo D.C. Wickner R.B. Science. 1995; 270: 93-95Google Scholar) and fibril formation in vitro (5Taylor K.L. Cheng N. Williams R.W. Steven A.C. Wickner R.B. Science. 1999; 283: 1339-1342Google Scholar, C. Bousset L. Komar A. S. J. Cullin C. Melki R. 2001; Scholar) are on the of the N-terminal The N-terminal prion domain is in the native C. Komar A.A. Bousset L. Fernandez-Bellot E. Cullin C. Melki R. J. Biol. Chem. 1999; 274: 13666-13674Google Scholar, S. Butler J. Mol. Biol. 1999; Scholar) and is in and residues of or of the has on the structure, or of Ure2 under a range of L. Zhang X.J. Wang L.Y. Zhou J.M. Perrett S. J. Mol. Biol. 2003; 328: 235-254Google Scholar, C. Bousset L. Komar A. S. J. Cullin C. Melki R. 2001; Scholar, S. Butler J. Mol. Biol. 1999; Scholar, Perrett S. J. Mol. Biol. 2002; Scholar, J.M. L. C. Perrett S. 2001; Scholar, L. H. M. Zhou J.M. Perrett S. 2003; Scholar). of is for a of human disease Biol. Sci. 1999; Scholar). The ability of or to by forming a structure is thought to cause disease M. Proc. Natl. Acad. Sci. U. S. A. 1994; Scholar, 2001; Scholar, E. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: Scholar, R. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: Scholar), by M. E. J. N. M. 2002; Scholar) or by of M. H. M. H. S. J. Science. 2001; Scholar, S. V. R. J. Mol. Biol. 2001; Scholar). is for and amyloid that the species to are or formed in the fibrillar C. H. 2001; Scholar, I. R. 2002; Scholar). ThT binding a to the of on the of amyloid formation L. Zhang X.J. Wang L.Y. Zhou J.M. Perrett S. J. Mol. Biol. 2003; 328: 235-254Google Scholar, V.N. J. Fink A.L. J. Biol. Chem. 2001; 276: Scholar). The time course of Ure2 amyloid formation monitored by ThT binding a a lag an growth and a plateau region (7Schlumpberger M. Wille H. Baldwin M.A. Butler D.A. Herskowitz I. Prusiner S.B. Protein Sci. 2000; 9: 440-451Google Scholar, 8Bousset L. Thomson N.H. Radford S.E. Melki R. EMBO J. 2002; 21: 2903-2911Google Scholar, 9Zhu L. Zhang X.J. Wang L.Y. Zhou J.M. Perrett S. J. Mol. Biol. 2003; 328: 235-254Google Scholar). The lag time be by with amyloid-like fibrils (5Taylor K.L. Cheng N. Williams R.W. Steven A.C. Wickner R.B. Science. 1999; 283: 1339-1342Google Scholar, 6Thual C. Komar A.A. Bousset L. Fernandez-Bellot E. Cullin C. Melki R. J. Biol. Chem. 1999; 274: 13666-13674Google Scholar, 7Schlumpberger M. Wille H. Baldwin M.A. Butler D.A. Herskowitz I. Prusiner S.B. Protein Sci. 2000; 9: 440-451Google Scholar) and is protein C. Komar A.A. Bousset L. Fernandez-Bellot E. Cullin C. Melki R. J. Biol. Chem. 1999; 274: 13666-13674Google Scholar, 9Zhu L. Zhang X.J. Wang L.Y. Zhou J.M. Perrett S. J. Mol. Biol. 2003; 328: 235-254Google Scholar). This is consistent with a 1993; Scholar, Biol. 2000; Scholar), the lag time the to of a of to form a stable or However, stable is growth the fibril is are a of that that characteristic in dye binding for Ure2 also to the appearance of formed amyloid-like by or (5Taylor K.L. Cheng N. Williams R.W. Steven A.C. Wickner R.B. Science. 1999; 283: 1339-1342Google Scholar, 6Thual C. Komar A.A. Bousset L. Fernandez-Bellot E. Cullin C. Melki R. J. Biol. Chem. 1999; 274: 13666-13674Google Scholar, 7Schlumpberger M. Wille H. Baldwin M.A. Butler D.A. Herskowitz I. Prusiner S.B. Protein Sci. 2000; 9: 440-451Google Scholar, 8Bousset L. Thomson N.H. Radford S.E. Melki R. EMBO J. 2002; 21: 2903-2911Google Scholar, 9Zhu L. Zhang X.J. Wang L.Y. Zhou J.M. Perrett S. J. Mol. Biol. 2003; 328: 235-254Google Scholar). particles or of Ure2 been observed by C. Komar A.A. Bousset L. Fernandez-Bellot E. Cullin C. Melki R. J. Biol. Chem. 1999; 274: 13666-13674Google Scholar) and (8Bousset L. Thomson N.H. Radford S.E. Melki R. EMBO J. 2002; 21: 2903-2911Google Scholar). However, the the time course of changes in ThT binding and of amyloid-like structure has been for Ure2. in that be observed in for In addition, the of species in fibril morphology which are apparent in images (8Bousset L. Thomson N.H. Radford S.E. Melki R. EMBO J. 2002; 21: 2903-2911Google Scholar, J. Protein Chem. Scholar, C. R. L.C. Fink A.L. Proc. Natl. Acad. Sci. U. S. A. 1999; Scholar). compare the time course of structural changes monitored by ThT binding and and the role of of the Ure2 on the of fibril and Ure2 and N-terminal and in with a and L. Zhang X.J. Wang L.Y. Zhou J.M. Perrett S. J. Mol. Biol. 2003; 328: 235-254Google Scholar, S. Butler J. Mol. Biol. 1999; Scholar). for the mutants S. Butler J. Mol. Biol. 1999; Scholar). at and in a to in or in L. Zhang X.J. Wang L.Y. Zhou J.M. Perrett S. J. Mol. Biol. 2003; 328: 235-254Google Scholar) and at for at to of amyloid formation of Ure2 monitored ThT binding fluorescence (7Schlumpberger M. Wille H. Baldwin M.A. Butler D.A. Herskowitz I. Prusiner S.B. Protein Sci. 2000; 9: 440-451Google Scholar, 9Zhu L. Zhang X.J. Wang L.Y. Zhou J.M. Perrett S. J. Mol. Biol. 2003; 328: 235-254Google Scholar, V.N. J. Fink A.L. J. Biol. Chem. 2001; 276: Scholar, H. M. Scholar). at a of or with L. Zhang X.J. Wang L.Y. Zhou J.M. Perrett S. J. Mol. Biol. 2003; 328: 235-254Google Scholar). at or to The range of used and the protein range for Ure2 and for Ure2 The in the are at the of for the of the of time the and for ThT L. Zhang X.J. Wang L.Y. Zhou J.M. Perrett S. J. Mol. Biol. 2003; 328: 235-254Google Scholar). in the time course of amyloid formation by ThT binding and the same of the protein on to for in with of for in a of a under conditions. with of used at a of the the to the force on the the of the of granular and fibrillar particles the with the compare the of the height of granular detected within a of for time the of of the height to the fibril a region the of height within an The height for fibril variation by to a The are the of the surface that the of particles or fibrils and that the with the that at the of the that are the surface by the process This has for the of the that formation fibril are and apparent the This in the increased to fibrils are in on the C. J. U. J. Mol. Biol. 1999; Scholar). Ure2 15Ure2 and Δ15–42Ure2 an in the by ThT binding a to the of on the of amyloid formation L. Zhang X.J. Wang L.Y. Zhou J.M. Perrett S. J. Mol. Biol. 2003; 328: 235-254Google Scholar, V.N. J. Fink A.L. J. Biol. Chem. 2001; 276: Scholar). We compared the of amyloid formation for Ure2 and a series of The mutants which the of which the Asn/Gln but the island of “normal” random within the which the residues of the and in which the Asn/Gln region has been The and Ure2 compared under a range of incubation temperatures and or and with or for a of are in The mutants and showed a in ThT binding fluorescence at protein This is consistent with the of residues for or of the prion in vivo D.C. Wickner R.B. Science. 1995; 270: 93-95Google Scholar, D.C. Wickner R.B. Proc. Natl. Acad. Sci. U. S. A. 1997; Scholar). the 15Ure2 showed a time course monitored by ThT to that for but with a lag time Δ15–42Ure2 showed reduced binding to ThT compared with 15Ure2 or However, under in at relatively protein the in ThT binding for Δ15–42Ure2 could be to be a lag time for Ure2 and to 15Ure2 at the same protein This a role of both Asn/Gln and of the in nucleation of amyloid However, also that of of the ability to form an amyloid-like of the Ure2 by height a to the morphology of at for J. Protein Chem. Scholar, C. R. L.C. Fink A.L. Proc. Natl. Acad. Sci. U. S. A. 1999; Scholar, C. J. U. J. Mol. Biol. 1999; Scholar, M. A. J. Biol. 1997; Scholar, 1999; Scholar, J. J. 2000; Scholar). the is and the surface is with an or on a The in height be by a to to form the characteristic of is that the of the to the of protein oligomers or fibrils results in an of the the height of the the surface be with and the height of by to be the by structural which the of the of and/or by the the height of the Ure2 dimer by a series of of Ure2 and the mutants Δ15–42Ure2 and in at the protein a to and is to be at protein S. Butler J. Mol. Biol. 1999; Scholar), species with corresponding to and are detected under at on the protein C. Komar A.A. Bousset L. Fernandez-Bellot E. Cullin C. Melki R. J. Biol. Chem. 1999; 274: 13666-13674Google Scholar). of a protein of or Ure2 a of particles The species has a height of 2.6 ± 0.5 The a of particles which in the range protein an at ± consistent with of the are observed for and that the to the height of the Ure2 or dimer by of of by ThT and the structural of the in detected by ThT binding for Ure2 and the mutants the time course of structural changes in by ThT binding and In the for a of the time course by the for Ure2 in at with formed fibrils are formed within detected by and the time course of ThT binding is highly and relatively L. Zhang X.J. Wang L.Y. Zhou J.M. Perrett S. J. Mol. Biol. 2003; 328: 235-254Google Scholar), that a plateau is within The Ure2 protein to a of of granular particles and in height to of the particles in The particles to be or after the of and are to represent aggregates. fibrillar are also detected in the The of a of or for fibril could for the of the lag time on Ure2 L. Zhang X.J. Wang L.Y. Zhou J.M. Perrett S. J. Mol. Biol. 2003; 328: 235-254Google Scholar). in the height of granular particles the time course of the by the of within a at time points the of incubation and the of fibril formation the lag phase region a in the of particles of height which by an in particles of height with an in ThT binding fluorescence the height of the Ure2 dimer of 2.6 ± 0.5 that the particles that during the lag phase are or the particles are to represent The late in the phase of ThT binding and a height of the observed for M. E. J. N. M. 2002; Scholar, C. H. 2001; Scholar, J. Protein Chem. Scholar, C. R. L.C. Fink A.L. Proc. Natl. Acad. Sci. U. S. A. 1999; Scholar, C. J. U. J. Mol. Biol. 1999; Scholar, 1999; Scholar, J. J. 2000; Scholar). are by fibrils and fibrils of fibrillar are detected ThT binding has a plateau and However, the of is in the is that both also and nm) to the of ThT of the fibril and morphology structural changes with variation in the ThT binding fluorescence in the plateau phase the of fibrils a height of and a relatively the height is observed to with a of to the fibrils after a of standing in at (8Bousset L. Thomson N.H. Radford S.E. Melki R. EMBO J. 2002; 21: 2903-2911Google Scholar). the course of the and of incubation at a in the fibril at the fibril the of fibril types the to a a height of ± for the fibril that in the plateau and a height of ± for the fibril that at incubation The that is apparent time is that fibrils but a or morphology that the of fibrils at time points fibrils (twisted fibrils by in of This that at least types of fibril are by their height and the of which The of the fibrils time that the fibrils be the fibrils by conversion or of the of for and a by ThT binding and that fibrils of Ure2 are by the of the growth phase are by plateau phase and at incubation in morphology time course a in the of fibril the appearance of and fibrils and The ThT binding that and to a are also to form amyloid-like structure, with a lag by of and fibrils at of the time course is in of the under with but with the protein of the mutants increased to a time course protein fibrils are observed for but only could be detected for The results that the observed in ThT binding for the with formation of fibrillar However, the fibrils are in and at incubation This is for which only However, the and fibrils or show a pattern of height changes with appearance of and height or fibrils ThT of the Δ15–42Ure2 the of ThT binding for the Δ15–42Ure2 in to with the of the fibrils to However, Ure2 and mutants by under at the that Ure2 is reduced under S. Butler J. Mol. Biol. 1999; Scholar), fibrils of Δ15–42Ure2 are readily formed on standing in at Ure2 fibrils and of formed at in or at used to Ure2 fibrils (8Bousset L. Thomson N.H. Radford S.E. Melki R. EMBO J. 2002; 21: 2903-2911Google Scholar, 13Baxa U. Speransky V. Steven A.C. Wickner R.B. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 5253-5260Google Scholar), are Δ15–42Ure2 fibrils and of with of and be and and or of the filaments be observed In the Δ15–42Ure2 fibrils are at time points In to the time course granular particles observed in that granular particles are in fibril formation and that the ThT binding under is the of the of and The the of ThT binding and the of fibrils for Ure2 and Δ15–42Ure2 could be the of a of Ure2 for the However, the of Ure2 fibrils under is also apparent L. Zhang X.J. Wang L.Y. Zhou J.M. Perrett S. J. Mol. Biol. 2003; 328: 235-254Google Scholar, C. Bousset L. Komar A. S. J. Cullin C. Melki R. 2001; Scholar). The for the ThT binding and the of fibrils for Δ15–42Ure2 is that the 15–42 region is for binding of the dye ThT. the of the Scholar, 2001; Scholar) that the region residues is relatively However, of residues and region This for the reduced binding of ThT to the ability to form fibrillar formed by Ure2. The of the Ure2 N-terminal domain in prion and has been by D.C. Wickner R.B. Science. 1995; 270: 93-95Google Scholar, D.C. Wickner R.B. Proc. Natl. Acad. Sci. U. S. A. 1997; Scholar), and the of the to form amyloid-like fibrils in vitro has also been demonstrated (5Taylor K.L. Cheng N. Williams R.W. Steven A.C. Wickner R.B. Science. 1999; 283: 1339-1342Google Scholar, C. Bousset L. Komar A. S. J. Cullin C. Melki R. 2001; Scholar). The mutants used in to the at the on the of D.C. Wickner R.B. Science. 1995; 270: 93-95Google Scholar, D.C. Wickner R.B. Proc. Natl. Acad. Sci. U. S. A. 1997; Scholar), the mutants used in at the protein on of the of the structure S. Butler J. Mol. Biol. 1999; and Protein of Ure2 in species of yeast has identified a conserved region in the corresponding to residues H. Wickner R.B. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: Scholar). Saccharomyces the highly conserved region extends residue to of region of the prion in prion strains and of the Ure2 protein in strains H. Wickner R.B. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: Scholar). This that the region residues is in for formation of the prion that the Ure2 mutants 15Ure2 and which within are to form amyloid-like fibrils is with the Ure2 that been in vivo that mutants be to or the prion for the properties of the conserved region in vivo a by the of with the prion of which the prion or the for a to maintain the prion which is by the Ure2 H. Wickner R.B. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: Scholar, H. Wickner R.B. Proc. Natl. Acad. Sci. U. S. A. 1999; Scholar). in vitro in the of for a and the of during to form amyloid-like of Ure2 mutants and in of the prion the is in a in an [URE3] prion of the Ure2 protein of a stable prion [URE3] the is in vivo in a of the [URE3] prion the is in a in an to the prion the is in vivo in a of the [URE3] prion the is in vivo in a is to N-terminal that the Ure2 region show the of Ure2 which is the of the [URE3] of the prion the is in a in an [URE3] prion H. Wickner R.B. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: Scholar, H. Wickner R.B. Proc. Natl. Acad. Sci. U. S. A. 1999; of the Ure2 protein of a stable prion [URE3] the is in vivo in a H. Wickner R.B. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: of the [URE3] prion the is in a in an D.C. Wickner R.B. Science. 1995; 270: 93-95Google to the prion the is in vivo in a D.C. Wickner R.B. Proc. Natl. Acad. Sci. U. S. A. 1997; of the [URE3] prion the is in vivo in a D.C. Wickner R.B. Proc. Natl. Acad. Sci. U. S. A. 1997; Scholar). is to N-terminal that the Ure2 region show the of Ure2 which is the of the [URE3] in a The increased lag time for formation of amyloid-like structure for the mutants in is consistent with a role of the conserved region of the Ure2 in forming or that the prion The of binding of the dye ThT to of fibril that the island of random within the is in formation of the structural motif or binding surface recognized by ThT. This the relatively of region which could also in and in vivo The fibril formation of the mutants compared with an for their increased in and in E. The in of and Ure2 in vitro is in S. Butler J. Mol. Biol. 1999; Scholar), which with of the native to to be in amyloid formation and L. Zhang X.J. Wang L.Y. Zhou J.M. Perrett S. J. Mol. Biol. 2003; 328: 235-254Google Scholar). In in morphology and Ure2 fibrils could be but to fibril In and fibrils in and Further, under the same series of fibrillar observed for and the This that the of of and Ure2 fibrils is the be to the properties of 15Ure2 and Δ15–42Ure2 in to of mutants in a could a has been demonstrated for the protein D.C. Wickner R.B. Proc. Natl. Acad. Sci. U. S. A. 1997; of Ure2 mutants in in E. or in in that be and that amyloid-like fibrils of Ure2 been observed in of of nucleation of amyloid-like structure, by ThT binding lag lag in ThT binding ThT binding in plateau phase by of fibrillar detected by after incubation in and that and fibrils show the same time-dependent changes in fibril under and that in fibril observed in and in E. S. Butler J. Mol. Biol. 1999; Scholar) or in L. Zhang X.J. Wang L.Y. Zhou J.M. Perrett S. J. Mol. Biol. 2003; 328: 235-254Google Scholar) in that be S. Butler J. Mol. Biol. 1999; Scholar), and that amyloid-like fibrils of Ure2 been observed in (7Schlumpberger M. Wille H. Baldwin M.A. Butler D.A. Herskowitz I. Prusiner S.B. Protein Sci. 2000; 9: 440-451Google of nucleation of amyloid-like structure, by ThT binding lag lag in ThT binding ThT binding in plateau phase of fibrillar detected by after incubation in and that and fibrils show the same time-dependent changes in fibril under and that in fibril observed in and in a of the time course of structural changes monitored by ThT binding and by for Ure2 that increased binding of ThT with appearance of fibrillar However, granular also observed under and also bind ThT. in the is the time appearance of a series of Ure2 fibril nm), fibrils nm), and types of fibrils and 9 nm), a of Consistent with this, fibrils a and or of fibrils could be This is to the results for M. E. J. N. M. 2002; Scholar, J. Protein Chem. Scholar, C. R. L.C. Fink A.L. Proc. Natl. Acad. Sci. U. S. A. 1999; Scholar, C. J. U. J. Mol. Biol. 1999; Scholar, 1999; Scholar, J. J. 2000; Scholar), S. V. R. 2002; Scholar). The of by of or to form a of fibril is a of fibril for D.A. Radford S.E. Thomson N.H. J. Mol. Biol. 2003; Scholar, R. C. M. J. L. M. L. Fink A.L. J. 2003; Scholar). The yeast prion has an N-terminal Asn/Gln Protein Chem. 2001; Scholar). Fibrils formed S. V. R. 2002; Scholar, M. R. Proc. Natl. Acad. Sci. U. S. A. 2001; Scholar, J. A. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: Scholar), Protein Chem. 2001; Scholar, M. R. Proc. Natl. Acad. Sci. U. S. A. 2001; Scholar, J. A. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: Scholar, S. 1997; Scholar), or Ure2 (5Taylor K.L. Cheng N. Williams R.W. Steven A.C. Wickner R.B. Science. 1999; 283: 1339-1342Google Scholar, 6Thual C. Komar A.A. Bousset L. Fernandez-Bellot E. Cullin C. Melki R. J. Biol. Chem. 1999; 274: 13666-13674Google Scholar, L. J. Melki R. J. Biol. 2003; Scholar) of the structural and properties of and unusual property of is the of native-like structural and properties within the amyloid-like fibrillar arrays (8Bousset L. Thomson N.H. Radford S.E. Melki R. EMBO J. 2002; 21: 2903-2911Google Scholar, 13Baxa U. Speransky V. Steven A.C. Wickner R.B. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 5253-5260Google Scholar). fibrils formed by incubation of show a fibrillar under S. 1997; Scholar), highly of the for Ure2 fibril (4Speransky V.V. Taylor K.L. Edskes H.K. Wickner R.B. Steven A.C. J. Cell Biol. 2001; 153: 1327-1335Google Scholar, K.L. Cheng N. Williams R.W. Steven A.C. Wickner R.B. Science. 1999; 283: 1339-1342Google Scholar, 13Baxa U. Speransky V. Steven A.C. Wickner R.B. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 5253-5260Google Scholar, U. Taylor K.L. Cheng N. Wickner R.B. Steven A.C. J. Biol. Chem. 2003; Scholar). results are consistent with a for Ure2 fibril in which the the and fibrils of the to be in a native-like of of the mutants 15Ure2 or Δ15–42Ure2 is to form amyloid-like fibrils Ure2 but with an increased lag amyloid forming This with the of residues of the Ure2 N-terminal in prion or in a in vivo D.C. Wickner R.B. Science. 1995; 270: 93-95Google Scholar, D.C. Wickner R.B. Proc. Natl. Acad. Sci. U. S. A. 1997; Scholar, H. Wickner R.B. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: Scholar). The of a series of fibrillar during of Ure2p fibrils the of a for fibril in native-like structure is within the fibrillar