Molecular chaperone Hsp104 can promote yeast prion generation

Abstract

[URE3] is an amyloid-based prion of Ure2p, a regulator of nitrogen catabolism in Saccharomyces cerevisiae. The Ure2p of the human pathogen Candida albicans can also be a prion in S. cerevisiae. We find that overproduction of the disaggregating chaperone, Hsp104, increases the frequency of de novo [URE3] prion formation by the Ure2p of S. cerevisiae and that of C. albicans. This stimulation is strongly dependent on the presence of the [PIN(+)] prion, known from previous work to enhance [URE3] prion generation. Our data suggest that transient Hsp104 overproduction enhances prion generation through persistent effects on Rnq1 amyloid, as well as during overproduction by disassembly of amorphous Ure2 aggregates (generated during Ure2p overproduction), driving the aggregation toward the amyloid pathway. Overproduction of other major cytosolic chaperones of the Hsp70 and Hsp40 families (Ssa1p, Sse1p, and Ydj1p) inhibit prion formation, whereas another yeast Hsp40, Sis1p, modulates the effects of Hsp104p on both prion induction and prion curing in a prion-specific manner. The same factor may both enhance de novo prion generation and destabilize existing prion variants, suggesting that prion variants may be selected by changes in the chaperone network.

Figure 1.—

Effects of different chaperones on de novo [URE3alb] prion induction. BY302 was transformed with both a chaperone overexpression plasmid and the P_GAL–URE2^alb plasmid. A total of 10⁵ cells were spread on −Ade plates to detect and compare [URE3alb] induction frequencies. At least three independent experiments were performed and average numbers were plotted. Error bars represent standard deviation.

Figure 2.—

Hsp104 can influence both [URE3] and [PSI⁺] prion induction. (A) Strain DK174 having the P_GAL–URE2 plasmid was transformed with either control vector or the HSP104 overexpression plasmid. After the standard induction procedure, 10⁶ cells were spread on −Ade plates. (B) A Similar induction assay was performed for the strain 74D-694 [psi⁻] [PIN⁺] having the P_GAL–SUP35 plasmid and either control vector or the HSP104 overexpression plasmid.

Figure 3.—

The presence of overproduced Hsp104p for a limited time creates long-lasting conditions for increased prion generation. (A) Scheme of the experiment. The upper line (I): Strain BY302 bearing the P_GAL–URE2^alb plasmid was transformed with the HSP104 overexpression plasmid. After growth for 30 generations the plasmid with HSP104 was lost and cells were grown further for 20 generations. An aliquot of cells was taken and the standard prion induction was performed; the rest of the culture was grown for 20 more generations and then also subjected to the prion induction procedure. The middle line (II): The same strain was subjected to prion induction after transformation with the HSP104 plasmid and growth for 30 generations. The bottom line (III): Prion induction was performed on the same strain without the HSP104 plasmid. (B) Western blotting with antiHsp104 antibodies showing that the level of Hsp104p returned to normal after the HSP104 plasmid was lost and cells were grown further for 20 generations. (C) Prion induction frequencies measured for I, II, and III. The frequency in I was indistinguishable when measured at 20 or 40 generations after the HSP104 plasmid was lost.

Figure 4.—

A model explaining the stimulatory effect of Hsp104p on prion induction. Hsp104p fragments Rnq1 amyloid and possibly Ure2 amyloid, creating new prion seeds. By modifying Rnq1 amyloid, Hsp104p may select out a different [PIN⁺] variant. In addition, it disassembles amorphous Ure2 aggregates, driving the aggregation toward the amyloid pathway.