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J. Biol. Chem., Vol. 282, Issue 16, 11931-11940, April 20, 2007

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Hsp40 Interacts Directly with the Native State of the Yeast Prion Protein Ure2 and Inhibits Formation of Amyloid-like Fibrils^*

Hui-Yong Lian, Hong Zhang, Zai-Rong Zhang, Harriët M. Loovers^¶, Gary W. Jones^¶1, Pamela J. E. Rowling^||, Laura S. Itzhaki^||, Jun-Mei Zhou, and Sarah Perrett²

From the National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, 15 Datun Road, Chaoyang District, Beijing 100101, China, the ^¶Department of Biology, National University of Ireland Maynooth, Maynooth, Ireland, the ^||Medical Research Council Cancer Cell Unit, Hutchison/Medical Research Council Research Centre, Hills Road, Cambridge CB2 0XZ, United Kingdom, and the Graduate School of the Chinese Academy of Sciences, Beijing 100039, China

Ure2 is the protein determinant of the [URE3] prion phenotype in Saccharomyces cerevisiae and consists of a flexible N-terminal prion-determining domain and a globular C-terminal glutathione transferase-like domain. Overexpression of the type I Hsp40 member Ydj1 in yeast cells has been found to result in the loss of [URE3]. However, the mechanism of prion curing by Ydj1 remains unclear. Here we tested the effect of overexpression of Hsp40 members Ydj1, Sis1, and Apj1 and also Hsp70 co-chaperones Cpr7, Cns1, Sti1, and Fes1 in vivo and found that only Ydj1 showed a strong curing effect on [URE3]. We also investigated the interaction of Ydj1 with Ure2 in vitro. We found that Ydj1 was able to suppress formation of amyloid-like fibrils of Ure2 by delaying the process of fibril formation, as monitored by thioflavin T binding and atomic force microscopy imaging. Controls using bovine serum albumin, Sis1, or the human Hsp40 homologues Hdj1 or Hdj2 showed no significant inhibitory effect. Ydj1 was only effective when added during the lag phase of fibril formation, suggesting that it interacts with Ure2 at an early stage in fibril formation and delays the nucleation process. Using surface plasmon resonance and size exclusion chromatography, we demonstrated a direct interaction between Ydj1 and both wild type and N-terminally truncated Ure2. In contrast, Hdj2, which did not suppress fibril formation, did not show this interaction. The results suggest that Ydj1 inhibits Ure2 fibril formation by binding to the native state of Ure2, thus delaying the onset of oligomerization.

Received for publication, July 19, 2006 , and in revised form, January 16, 2007.

^* This work was supported by Natural Science Foundation of China Grants 30470363, 30620130109, and 30670428; Chinese Ministry of Science and Technology Grants 2006CB500703 and 2006CB910903; Chinese Academy of Sciences Knowledge Innovation Project Grant KSCX2-SW214-3; and core funding from the Institute of Biophysics (to S. P.). Work performed in the Itzhaki laboratory was supported by the Medical Research Council (to L. S. I.) and by a European Molecular Biology Organization short term fellowship (to S. P.). Exchange between the Jones and Perrett laboratories was supported by China/Ireland Science and Technology Collaboration Research Fund Grant CI-2004-01. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Fig. S1.

¹ Supported by the Irish Health Research Board.

² To whom correspondence should be addressed. Tel.: 86-10-6485-6727; Fax: 86-10-6487-2026; E-mail: sarah.perrett{at}iname.com.

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