Molecular Chaperones and the Assembly of the Prion Ure2p in Vitro

Abstract

The protein Ure2 from Saccharomyces cerevisiae possesses prion properties at the origin of the [URE3] trait. In vivo, a high molecular weight form of inactive Ure2p is associated to [URE3]. The faithful and continued propagation of [URE3] is dependent on the expression levels of molecular chaperones from the Hsp100, -70, and -40 families; however, so far, their role is not fully documented. Here we investigate the effects of molecular chaperones from the Hsp40, Hsp70, Hsp90, and Hsp100 families and the chaperonin CCT/Tric on the assembly of full-length Ure2p. We show that Hsp104p greatly stimulates Ure2p aggregation, whereas Ssa1p, Ydj1p, Sis1p, and Hsp82p inhibit aggregation to different extents. The nature of the high molecular weight Ure2p species that forms in the presence of the different molecular chaperones and their nucleotide dependence is described. We show that Hsp104p favors the aggregation of Ure2p into non-fibrillar high molecular weight particles, whereas Ssa1p, Ydj1p, Sis1p, and Hsp82p sequester Ure2p in spherical oligomers. Using fluorescently labeled fulllength Ure2p and Ure2p-(94 -354) and fluorescence polarization, we show that Ssa1p binding to Ure2p is ATP-dependent, whereas that of Hsp104p is not. We also show that Ssa1p preferentially interacts with the N-terminal domain of Ure2p that is critical for prion propagation, whereas Ydj1p preferentially interacts with the C-terminal domain of the protein, and we discuss the significance of this observation. Finally, the affinities of Ssa1p, Ydj1p, and Hsp104p for Ure2p are determined. Our in vitro observations bring new insight into the mechanism by which molecular chaperones influence the propagation of [URE3].The [URE3] trait, discovered in the early 70s (1), is an inheritable prion factor in the yeast Saccharomyces cerevisiae (2). In a manner similar to what is observed for the vertebrate prion PrP, the [URE3] prion state is associated with a change in the solubility of the protein Ure2 (3).Similarly to other proteins with prion properties from S. cerevisiae, Ure2p is a two-domain protein. The physical boundary between the two domains of this 354-amino acid polypeptide is amino acid residue 94 (4). The asparagine-, glutamine-, serine-, and threonine-rich N-terminal domain of Ure2p (62% of amino acid residues) is crucial for prion propagation and flexible (4, 5, 6), whereas its C-terminal domain is compactly folded and mainly ␣-helical (7, 8). The latter domain binds glutathione (9), has glutathione peroxidase activity (10), and is sufficient to regulate nitrogen metabolism in bakers' yeast cells (11)(12)(13)(14).In vitro, at neutral pH, soluble Ure2p spontaneously forms long, twisted fibrils (4,(15)(16)(17). It is widely believed that similar fibrils form in vivo and are at the origin of the [URE3] trait (18).In vivo, the continued propagation of yeast prions is highly dependent on the expression levels of a number of molecular chaperones. Indeed, members of the Hsp100, Hsp70, and (20). It is worth noting that in vivo, the effect of a molecu...

Single methyl group determines prion propagation and protein degradation activities of yeast heat shock protein (Hsp)-70 chaperones Ssa1p and Ssa2p

“…When purified, Ure2p spontaneously assembles into amyloid filaments, and this assembly is inhibited by addition of purified Ssa1p (23,24). In our hands assembly of purified Ure2p into amyloid showed a sigmoidal curve with a very short lag phase of about 5 min (Fig.…”

Curing of Yeast [URE3] Prion by the Hsp40 Cochaperone Ydj1p Is Mediated by Hsp70

“…In vitro, both Ydj1p and Ssa1p inhibit Ure2p amyloid formation (Lian et al 2007;Savistchenko et al 2008). These latter observations suggest that Hsp70 and Hsp40 each interact with Ure2p in vivo, and it has been proposed that Ydj1p and Ssa1p cure [URE3] by binding Ure2p and inhibiting its ability to form amyloid in cells (Lian et al 2007;Savistchenko et al 2008). Such a mechanism has not been confirmed experimentally.…”

“…Overexpressing Ssa1p, but not the nearly identical Ssa2p, antagonizes [URE3], and cells lacking both Ssa1p and Ssa2p cannot propagate [URE3] (Schwimmer and Masison 2002;Sharma and Masison 2008). In vitro, both Ydj1p and Ssa1p inhibit Ure2p amyloid formation (Lian et al 2007;Savistchenko et al 2008). These latter observations suggest that Hsp70 and Hsp40 each interact with Ure2p in vivo, and it has been proposed that Ydj1p and Ssa1p cure [URE3] by binding Ure2p and inhibiting its ability to form amyloid in cells (Lian et al 2007;Savistchenko et al 2008).…”

Molecular Chaperone Hsp104 Can Promote Yeast Prion Generation

“…Consistent with this hypothesis, several chaperones influence the rate of spontaneous assembly of both Sup35p and Ure2p into amyloid-like fibers in vitro. Members of Hsp70 and Hsp40 families were reported to reduce the polymerization rate for both proteins (Krzewska and Melki 2006;Savistchenko et al 2008;Shorter and Lindquist 2008). In contrast, Hsp104p was the only factor reported to promote amyloid fibrillization in vitro of both Sup35p and Ure2p (Krzewska and Melki 2006;Shorter and Lindquist 2006).…”