Influence of specific HSP70 domains on fibril formation of the yeast prion protein Ure2


Xu, Li-Qiong and Wu, Si and Buell, Alexander K. and Cohen, Samuel I.A. and Chen, Li-Jun and Hu, Wan-Hui and Cusack, Sarah and Itzhaki, Laura S. and Zhang, Hong and Knowles, Tuomas P.J. and Dobson, Christopher M. and Welland, Mark E. and Jones, Gary W. and Perrett, Sarah (2013) Influence of specific HSP70 domains on fibril formation of the yeast prion protein Ure2. Philosophical Transactions of the Royal Society B, 368 (1617). p. 20110410. ISSN 0962-8436

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Abstract

Ure2p is the protein determinant of the Saccharomyces cerevisiae prion state [URE3]. Constitutive overexpression of the HSP70 family member SSA1 cures cells of [URE3]. Here, we show that SSA1 increases the lag time of Ure2p fibril formation in vitro in the presence or absence of nucleotide. The presence of the HSP40 co-chaperone Ydj1p has an additive effect on the inhibition of Ure2p fibril formation, whereas the Ydj1p H34Q mutant shows reduced inhibition alone and in combination with Ssa1p. In order to investigate the structural basis of these effects, we constructed and tested an Ssa1p mutant lacking the ATPase domain, as well as a series of C-terminal truncation mutants. The results indicate that Ssa1p can bind to Ure2p and delay fibril formation even in the absence of the ATPase domain, but interaction of Ure2p with the substrate-binding domain is strongly influenced by the C-terminal lid region. Dynamic light scattering, quartz crystal microbalance assays, pull-down assays and kinetic analysis indicate that Ssa1p interacts with both native Ure2p and fibril seeds, and reduces the rate of Ure2p fibril elongation in a concentration-dependent manner. These results provide new insights into the structural and mechanistic basis for inhibition of Ure2p fibril formation by Ssa1p and Ydj1p.

Item Type: Article
Additional Information: © 2013 The Authors. Published by the Royal Society under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0/, which permits unrestricted use, provided the original author and source are credited.
Keywords: prion; amyloid; Ure2p; Ssa1p; chaperone; quartz crystal microbalance;
Academic Unit: Faculty of Science and Engineering > Biology
Item ID: 6859
Identification Number: 10.1098/rstb.2011.0410
Depositing User: Dr. Gary Jones
Date Deposited: 19 Jan 2016 15:29
Journal or Publication Title: Philosophical Transactions of the Royal Society B
Publisher: The Royal Society
Refereed: Yes
Funders: Chinese Ministry of Science and Technology, National Natural Science Foundation of China, Chinese Academy of Sciences, Royal Society, Wellcome Trust, Magdalene College, Cambridge, St John’s College, Cambridge, Schiff Foundation, Science Foundation Ireland (SFI)
URI:

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