title: Influence of specific HSP70 domains on fibril formation of the yeast prion protein Ure2.
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 Ssa1p 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.
authors.affiliation: National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, , 15 Datun Road, Chaoyang, Beijing 100101, People's Republic of China.
authors.forename: Li-Qiong
authors.initials: LQ
authors.lastname: Xu
authors.forename: Si
authors.initials: S
authors.lastname: Wu
authors.forename: Alexander K
authors.initials: AK
authors.lastname: Buell
authors.forename: Samuel I A
authors.initials: SI
authors.lastname: Cohen
authors.forename: Li-Jun
authors.initials: LJ
authors.lastname: Chen
authors.forename: Wan-Hui
authors.initials: WH
authors.lastname: Hu
authors.forename: Sarah A
authors.initials: SA
authors.lastname: Cusack
authors.forename: Laura S
authors.initials: LS
authors.lastname: Itzhaki
authors.forename: Hong
authors.initials: H
authors.lastname: Zhang
authors.forename: Tuomas P J
authors.initials: TP
authors.lastname: Knowles
authors.forename: Christopher M
authors.initials: CM
authors.lastname: Dobson
authors.forename: Mark E
authors.initials: ME
authors.lastname: Welland
authors.forename: Gary W
authors.initials: GW
authors.lastname: Jones
authors.forename: Sarah
authors.initials: S
authors.lastname: Perrett
mesh_terms: D000251:Adenosine Triphosphatases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D015374:Biosensing Techniques; D015966:Gene Expression Regulation, Fungal; D005979:Glutathione Peroxidase / Q000235:genetics / Q000378:metabolism*; D018840:HSP70 Heat-Shock Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D008958:Models, Molecular; D018832:Molecular Chaperones; D011328:Prions / Q000235:genetics / Q000378:metabolism*; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D018840:HSP70 Heat-Shock Proteins; D018832:Molecular Chaperones; D011328:Prions; D029701:Saccharomyces cerevisiae Proteins; D005979:Glutathione Peroxidase; C067020:URE2 protein, S cerevisiae; D000251:Adenosine Triphosphatases; C095973:SSA1 protein, S cerevisiae