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.
Highlights
Prions are infectious proteins, accounting for a group of invariably fatal neurodegenerative diseases in mammals, including Creutzfeldt –Jakob disease in humans, and bovine spongiform encephalopathy and scrapie in animals [1]
As the ATPase activity of Ssa1p is stimulated by the cochaperone Ydj1p [39,42], we examined the effect of nucleotide on inhibition of Ure2p fibril formation by Ssa1p in the presence and absence of Ydj1p
Our results show that the nucleotide-binding domain (NBD) and the C-terminal random coil regions of Ssa1p do not contribute to inhibition of Ure2p fibril formation, whereas further truncation of the C-terminal region reduces the inhibition ability of Ssa1p, with helices C and D of the a-helix bundle each contributing to the inhibition effect
Summary
Prions are infectious proteins, accounting for a group of invariably fatal neurodegenerative diseases in mammals, including Creutzfeldt –Jakob disease in humans, and bovine spongiform encephalopathy and scrapie in animals [1]. Certain mutants of DnaK can refold luciferase normally in the absence of any significant ATP turnover, and complement a DdnaK strain of Escherichia coli This indicates that removal of the ATPase function of HSP70 does not necessarily ablate its ability to protect proteins from misfolding and aggregation in vitro and in vivo [49]. A 100 ml mixture of 10 mM Ure2p seeds and 20 mM wild-type (WT) or DA Ssa1p was incubated in Tris buffer pH 8.4 in a 308C water bath for 2 h and loaded onto the surface of 0.9 ml of a 40 per cent sucrose solution prior to centrifugation at 61 000 rpm for 30 min in a S140AT rotor at 48C using a Hitachi CS150GXL centrifuge. The pellets were washed twice and resuspended in Tris buffer pH 8.4 containing 1 per cent sodium dodecyl sulfate (SDS), and boiled for 10 min before applying to the SDS-polyacrylamide gel electrophoresis (PAGE)
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