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Licensed Unlicensed Requires Authentication Published by De Gruyter August 8, 2005

Characterization of oligomeric species in the fibrillization pathway of the yeast prion Ure2p

Silvia Catharino , Johannes Buchner and Stefan Walter
From the journal

Abstract

The [URE3] prion of Saccharomyces cerevisiae shares many features with mammalian prions and poly-glutamine related disorders and has become a model for studying amyloid diseases. The development of the [URE3] phenotype is thought to be caused by a structural switch in the Ure2p protein. In [URE3] cells, Ure2p is found predominantly in an aggregated state, while it is a soluble dimer in wild-type cells. In vitro, Ure2p forms fibrils with amyloid-like properties. Several studies suggest that the N-terminal domain of Ure2p is essential for prion formation. In this work, we investigated the fibril formation of Ure2p by isolating soluble oligomeric species, which are generated during fibrillization, and characterized them with respect to size and structure. Our data support the critical role of the N-terminal domain for fibril formation, as we observed fibrils in the presence of 5 M guanidinium chloride, conditions at which the C-terminal domain is completely unfolded. Based on fluorescence measurements, we conclude that the structure of the C-terminal domain is very similar in dimeric and fibrillar Ure2p. When studying the time course of fibrillization, we detected the formation of small, soluble oligomeric species during the early stages of the process. Their remarkable resistance against denaturants, their increased content of β-structure, and their ability to ‘seed’ Ure2p fibrillization suggest that conversion to the amyloid-like conformation has already occurred. Thus, they likely represent critical intermediates in the fibrillization pathway of Ure2p.

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References

Balbirnie, M., Grothe, R., and Eisenberg, D.S. (2001). An amyloid-forming peptide from the yeast prion Sup35 reveals a dehydrated beta-sheet structure for amyloid. Proc. Natl. Acad. Sci. USA98, 2375–2380.10.1073/pnas.041617698Search in Google Scholar

Baxa, U., Speransky, V., Steven, A.C., and Wickner, R.B. (2002). Mechanism of inactivation on prion conversion of the Saccharomyces cerevisiae Ure2 protein. Proc. Natl. Acad. Sci. USA99, 5253–5260.10.1073/pnas.082097899Search in Google Scholar

Baxa, U., Taylor, K.L., Wall, J.S., Simon, M.N., Cheng, N., Wickner, R.B., and Steven, A.C. (2003). Architecture of Ure2p prion filaments: the N-terminal domains form a central core fiber. J. Biol. Chem.278, 43717–43727.10.1074/jbc.M306004200Search in Google Scholar

Beck, T. and Hall, M.N. (1999). The TOR signalling pathway controls nuclear localization of nutrient-regulated transcription factors. Nature402, 689–692.10.1038/45287Search in Google Scholar

Blinder, D., Coschigano, P.W., and Magasanik, B. (1996). Interaction of the GATA factor Gln3p with the nitrogen regulator Ure2p in Saccharomyces cerevisiae. J. Bacteriol.178, 4734–4736.10.1128/jb.178.15.4734-4736.1996Search in Google Scholar

Boehm, G. (1997). CDNN: CD Spectra Deconvolution, Version 2.1.Search in Google Scholar

Bousset, L., Belrhali, H., Janin, J., Melki, R., and Morera, S. (2001a). Structure of the globular region of the prion protein Ure2 from the yeast Saccharomyces cerevisiae. Structure9, 39–46.10.1016/S0969-2126(00)00553-0Search in Google Scholar

Bousset, L., Belrhali, H., Melki, R., and Morera, S. (2001b). Crystal structures of the yeast prion Ure2p functional region in complex with glutathione and related compounds. Biochemistry40, 13564–13573.10.1021/bi011007bSearch in Google Scholar

Bousset, L., Thomson, N.H., Radford, S.E., and Melki, R. (2002). The yeast prion Ure2p retains its native alpha-helical conformation upon assembly into protein fibrils in vitro. EMBO J.21, 2903–2911.10.1093/emboj/cdf303Search in Google Scholar

Chernoff, Y.O., Derkach, I.L., and Inge-Vechtomov, S.G. (1993). Multicopy SUP35 gene induces de-novo appearance of psi-like factors in the yeast Saccharomyces cerevisiae. Curr. Genet.24, 268–270.10.1007/BF00351802Search in Google Scholar

Derkatch, I.L., Bradley, M.E., Hong, J.Y., and Liebman, S.W. (2001). Prions affect the appearance of other prions: the story of [PIN(+)]. Cell106, 171–182.10.1016/S0092-8674(01)00427-5Search in Google Scholar

Edskes, H.K., Gray, V.T., and Wickner, R.B. (1999). The [URE3] prion is an aggregated form of Ure2p that can be cured by overexpression of Ure2p fragments. Proc. Natl. Acad. Sci. USA96, 1498–1503.10.1073/pnas.96.4.1498Search in Google Scholar

Fernandez-Bellot, E., Guillemet, E., Ness, F., Baudin-Baillieu, A., Ripaud, L., Tuite, M., and Cullin, C. (2002). The [URE3] phenotype: evidence for a soluble prion in yeast. EMBO Rep.3, 76–81.10.1093/embo-reports/kvf011Search in Google Scholar

Gill, S.C. and von Hippel, P.H. (1989). Calculation of protein extinction coefficients from amino acid sequence data. Anal. Biochem.182, 319–326.10.1016/0003-2697(89)90602-7Search in Google Scholar

Ionescu-Zanetti, C., Khurana, R., Gillespie, J.R., Petrick, J.S., Trabachino, L.C., Minert, L.J., Carter, S.A., and Fink, A.L. (1999). Monitoring the assembly of Ig light-chain amyloid fibrils by atomic force microscopy. Proc. Natl. Acad. Sci. USA96, 13175–13179.10.1073/pnas.96.23.13175Search in Google Scholar

Jiang, Y., Li, H., Zhu, L., Zhou, J.M., and Perrett, S. (2004). Amyloid nucleation and hierarchical assembly of Ure2p fibrils. Role of asparagine/glutamine repeat and nonrepeat regions of the prion domains. J. Biol. Chem.279, 3361–3369.10.1074/jbc.M310494200Search in Google Scholar

Kayed, R., Head, E., Thompson, J.L., McIntire, T.M., Milton, S.C., Cotman, C.W., and Glabe, C.G. (2003). Common structure of soluble amyloid oligomers implies common mechanism of pathogenesis. Science300, 486–489.10.1126/science.1079469Search in Google Scholar

Komar, A.A., Lesnik, T., Cullin, C., Guillemet, E., Ehrlich, R., and Reiss, C. (1997). Differential resistance to proteinase K digestion of the yeast prion-like (Ure2p) protein synthesized in vitro in wheat germ extract and rabbit reticulocyte lysate cell-free translation systems. FEBS Lett.415, 6–10.10.1016/S0014-5793(97)01082-XSearch in Google Scholar

Komar, A.A., Melki, R., and Cullin, C. (1999). The [URE3] yeast prion: from genetics to biochemistry. Biochemistry (Moscow)64, 1401–1407.Search in Google Scholar

LeVine, H. (1993). Thioflavine T interaction with synthetic Alzheimer's disease β-amyloid peptides: detection of amyloid aggregation in solution. Protein Sci.2, 404–410.10.1002/pro.5560020312Search in Google Scholar PubMed PubMed Central

Lindquist, S., Krobitsch, S., Li, L., and Sondheimer, N. (2001). Investigating protein conformation-based inheritance and disease in yeast. Philos. Trans. R. Soc. Lond. B Biol. Sci.356, 169–176.10.1098/rstb.2000.0762Search in Google Scholar PubMed PubMed Central

Masison, D.C., Maddelein, M.L., and Wickner, R.B. (1997). The prion model for [URE3] of yeast: spontaneous generation and requirements for propagation. Proc. Natl. Acad. Sci. USA94, 12503–12508.10.1073/pnas.94.23.12503Search in Google Scholar PubMed PubMed Central

Patino, M.M., Liu, J.J., Glover, J.R., and Lindquist, S. (1996). Support for the prion hypothesis for inheritance of a phenotypic trait in yeast. Science273, 622–626.10.1126/science.273.5275.622Search in Google Scholar

Perrett, S., Freeman, S.J., Butler, P.J., and Fersht, A.R. (1999). Equilibrium folding properties of the yeast prion protein determinant Ure2. J. Mol. Biol.290, 331–345.10.1006/jmbi.1999.2872Search in Google Scholar

Petkova, A.T., Leapman, R.D., Guo, Z., Yau, W.M., Mattson, M.P., and Tycko, R. (2005). Self-propagating, molecular-level polymorphism in Alzheimer's β-amyloid fibrils. Science307, 262–265.10.1126/science.1105850Search in Google Scholar

Riek, R., Hornemann, S., Wider, G., Billeter, M., Glockshuber, R., and Wuthrich, K. (1996). NMR structure of the mouse prion protein domain PrP(121–321). Nature382, 180–182.10.1038/382180a0Search in Google Scholar

Schneider, S.W., Larmer, J., Henderson, R.M., and Oberleithner, H. (1998). Molecular weights of individual proteins correlate with molecular volumes measured by atomic force microscopy. Pflüger's Arch.435, 362–367.10.1007/s004240050524Search in Google Scholar

Speransky, V.V., Taylor, K.L., Edskes, H.K., Wickner, R.B., and Steven, A.C. (2001). Prion filament networks in [URE3] cells of Saccharomyces cerevisiae. J. Cell Biol.153, 1327–1336.10.1083/jcb.153.6.1327Search in Google Scholar

Thual, C., Komar, A.A., Bousset, L., Fernandez-Bellot, E., Cullin, C., and Melki, R. (1999). Structural characterization of Saccharomyces cerevisiae prion-like protein Ure2. J. Biol. Chem.274, 13666–13674.10.1074/jbc.274.19.13666Search in Google Scholar

Thual, C., Bousset, L., Komar, A.A., Walter, S., Buchner, J., Cullin, C., and Melki, R. (2001). Stability, folding, dimerization, and assembly properties of the yeast prion Ure2p. Biochemistry40, 1764–1773.10.1021/bi001916lSearch in Google Scholar

Tuite, M.F. (2000). Yeast prions and their prion-forming domain. Cell100, 289–292.10.1016/S0092-8674(00)80663-7Search in Google Scholar

Umland, T.C., Taylor, K.L., Rhee, S., Wickner, R.B., and Davies, D.R. (2001). The crystal structure of the nitrogen regulation fragment of the yeast prion protein Ure2p. Proc. Natl. Acad. Sci. USA98, 1459–1464.10.1073/pnas.98.4.1459Search in Google Scholar PubMed PubMed Central

Uptain, S.M. and Lindquist, S. (2002). Prions as protein-based genetic elements. Annu. Rev. Microbiol.56, 703–741.10.1146/annurev.micro.56.013002.100603Search in Google Scholar

Valle, F., DeRose, J.A., Dietler, G., Kawe, M., Pluckthun, A., and Semenza, G. (2002). AFM structural study of the molecular chaperone GroEL and its two-dimensional crystals: an ideal ‘living’ calibration sample. Ultramicroscopy93, 83–89.10.1016/S0304-3991(02)00149-3Search in Google Scholar

Wickner, R.B. (1994). [URE3] as an altered URE2 protein: evidence for a prion analog in Saccharomyces cerevisiae. Science264, 566–569.10.1126/science.7909170Search in Google Scholar

Wickner, R.B., Masison, D.C., and Edskes, H.K. (1995). [PSI] and [URE3] as yeast prions. Yeast11, 1671–1685.10.1002/yea.320111609Search in Google Scholar

Wickner, R.B., Taylor, K.L., Edskes, H.K., Maddelein, M.L., Moriyama, H., and Roberts, B.T. (1999). Prions in Saccharomyces and Podospora spp.: protein-based inheritance. Microbiol. Mol. Biol. Rev.63, 844–861.10.1128/MMBR.63.4.844-861.1999Search in Google Scholar

Zhu, L., Zhang, X.J., Wang, L.Y., Zhou, J.M., and Perrett, S. (2003). Relationship between stability of folding intermediates and amyloid formation for the yeast prion Ure2p: a quantitative analysis of the effects of pH and buffer system. J. Mol. Biol.328, 235–254.10.1016/S0022-2836(03)00249-3Search in Google Scholar

Published Online: 2005-08-08
Published in Print: 2005-07-01

©2005 by Walter de Gruyter Berlin New York

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