Influence of prion variant and yeast strain variation on prion-molecular chaperone requirements

Abstract

Prions of budding yeast serve as a tractable model of amyloid behavior. Here we address the issue of the effect of yeast strain variation on prion stability, focusing also on the effect of amyloid conformation and the involvement of the co-chaperone Sis1, an essential J-protein partner of Hsp70. We found, despite an initial report to the contrary, that yeast strain background has little effect on the requirement for particular Sis1 domains for stable propagation of the prion [RNQ+], if the level of Sis1 expression is controlled. On the other hand, some variation in prion behavior was observed between yeast strains, in particular, the stability of certain [PSI+] variants. Future examination of such yeast strain-specific phenomena may provide useful insights into the basis of prion/chaperone dynamics.

Figure 1

Maintenance of [RNQ⁺] by Sis1 expression constructs. (A) Gene structure diagrams of Sis1 expression constructs. Gene regions are denoted using the following notation: J, J-domain; G/F, glycine/phenylalanine-rich region; G/M, glycine/methionine-rich region; peptide-binding, C-terminal peptide-binding domains I and II. Dashed line indicates a region has been deleted. (B) Plasmid shuffling experiments to test the domain requirements of Sis1 by [RNQ⁺]. Plasmids expressing Sis1 constructs illustrated in (A). Detergent resistant Rnq1 aggregates indicative of the presence of [RNQ⁺] were visualized by SDDAGE followed by immunoblot analysis. Control wild-type [RNQ⁺] and [rnq⁻] cells were included for comparison. Dotted lines separate lanes taken from different parts of the same gel. (C) Sis1 protein expression levels in yeast strains 74D-694 (74D) and W303 (W303). Cell lysates from wild-type or sis1-Δ cells expressing Sis1–21 from a plasmid were subjected to SDS-PAGE followed by immunoblotting with a Sis1-specific antibody. Antibody specific for Ssc1 was used as a loading control.

Figure 2

Loss of three distinct strong [PSI⁺] variants in the W303 genetic background upon Sis1 repression. (A and B) Time course of SIS1-repression of [PSI⁺]^SC4 cells. [PSI⁺]^SC4 cells were harvested after the indicated number of generations of growth in the presence of doxycycline and plated onto rich media. The fraction of cells remaining [PSI⁺]^SC4 (pink) vs. [psi⁻] (red) was (A) determined and (B) plotted (SC4, ■). The solid grey line represents a best-fit line through the data. (C) Lysates from sis1-Δ [TETrSIS1], [PSI⁺]^SC4 cells at various generations after addition of doxycycline, and from [psi⁻] control cells, were resolved by SDDAGE (top part) and SDS-PAGE (bottom parts). Sup35 and Sis1 were visualized by immunoblotting with Sup35- and Sis1-specific antibodies. Antibody specific for Ssc1 was used as a loading control. (D and E) Time courses of SIS1-repression in cells bearing strong [PSI⁺] variants [PSI⁺]^VH (VH, ●) and [PSI⁺]^STR (STR, ◆). Time courses were conducted as described in (A and B). Grey line is the fit curve from (B) shown for comparison.

Figure 3

Kinetics of loss of strong and weak [PSI⁺] variants in the 74D-694 genetic background upon Sis1 repression. (A) Time course of SIS1-repression of strong variant-bearing cells. 74D-694 cells bearing strong [PSI⁺] variants [PSI⁺]^SC4 (○) and [PSI⁺]^STR (◊) were harvested after the indicated number of generations of growth in the presence of doxycycline and plated onto rich media. The fraction of cells remaining [PSI⁺] based on colony color was plotted. For comparison, data for [PSI⁺]^SC4 in the W303 strain background, which also appears in Figure 2 is shown (■). The solid grey line represents a best fit line through the data. (B) Weak [PSI⁺] variants [PSI⁺]^SC37 (●) and [PSI⁺]^VL (◆), as in (A). The grey line is the best fit line for strong strains from (A) shown for comparison (note: change in scale of the x-axis).

Figure 4

Curing of three distinct weak [PSI⁺] variants upon Sis1 repression in the W303 genetic background. (A and B) Time course of SIS1-repression of [PSI⁺]^SC37 cells of the W303 background. [PSI⁺]^SC37 cells were harvested after the indicated number of generations of growth in the presence of doxycycline and plated onto rich media. The fraction of cells remaining [PSI⁺]^SC37 (pink) vs. [psi⁻] (red) was (A) determined and (B) plotted (SC37, ●). Dashed line represents a trend line connecting data points. (C) Resolution of prion aggregates by SDDAGE. Lysates from sis1-Δ [TETrSIS1], [PSI⁺]^SC37 cells at various generations after addition of doxycycline, and from [psi-] control cells, were resolved by SDDAGE (top part) and SDS-PAGE (bottom parts). Sup35 and Sis1 were visualized by immunoblotting with Sup35- and Sis1-specific antibodies. Antibody specific for Ssc1 was used as a loading control. (D) Time courses of SIS1-repression in cells bearing weak [PSI⁺] variants [PSI⁺]^VL (VL, ◆) and [PSI⁺]^VK (VK, ■). Time courses were conducted as described in (A and B). Dashed line represents a trend line connecting data points.

Figure 5

Unusual curing kinetics of weak [PSI⁺] strains in W303 are not due to “cryptic” Sis1-independent [PSI⁺] variants. (A and B) SIS1 repression experiments described in the legend to Figure 4 were repeated using (A) the initial cell population (Fig. 4C, first lane) and (B) two individual [PSI⁺]^SC37 colonies isolated after 64 generations of Sis1 repression (Fig. 4C, last lane).