Investigating the interactions of yeast prions: [SWI+], [PSI+], and [PIN+]

Abstract

Multiple prion elements, which are transmitted as heritable protein conformations and often linked to distinct phenotypes, have been identified in the budding yeast, Saccharomyces cerevisiae. It has been shown that overproduction of a prion protein Swi1 can promote the de novo conversion of another yeast prion [PSI(+)] when Sup35 is co-overproduced. However, the mechanism underlying this Pin(+) ([PSI(+)] inducible) activity is not clear. Moreover, how the Swi1 prion ([SWI(+)]) interacts with other yeast prions is unknown. Here, we demonstrate that the Pin(+) activity associated with Swi1 overproduction is independent of Rnq1 expression or [PIN(+)] conversion. We also show that [SWI(+)] enhances the appearance of [PSI(+)] and [PIN(+)]. However, [SWI(+)] significantly compromises the Pin(+) activity of [PIN(+)] when they coexist. We further demonstrate that a single yeast cell can harbor three prions, [PSI(+)], [PIN(+)], and [SWI(+)], simultaneously. However, under this condition, [SWI(+)] is significantly destabilized. While the propensity to aggregate underlies prionogenesis, Swi1 and Rnq1 aggregates resulting from overproduction are usually nonheritable. Conversely, prion protein aggregates formed in nonoverexpressing conditions or induced by preexisting prion(s) are more prionogenic. For [PSI(+)] and [PIN(+)] de novo formation, heterologous "facilitators," such as preexisting [SWI(+)] aggregates, colocalize only with the newly formed ring-/rod-shaped Sup35 or Rnq1 aggregates, but not with the dot-shaped mature prion aggregates. Their colocalization frequency is coordinated with their prion inducibility, indicating that prion-prion interactions mainly occur at the early initiation stage. Our results provide supportive evidence for the cross-seeding model of prionogenesis and highlight a complex interaction network among prions in yeast.

Keywords: Saccharomyces cerevisiae; [SWI+]; prion interactions; prionogenesis; protein aggregation.

Figure 1

The effects of Swi1 overproduction and [SWI⁺] on de novo [PSI⁺] formation. (A) Sup35NM-GFP (from p413CUP1–NMGFP) and one of the indicated proteins were co-overproduced in a [pin⁻] or rnq1Δ strain to induce [PSI⁺]. Overproduction of Swi1 (Swi1↑), Ure2₁–₆₅–GFP (Ure2↑), polyQ103–GFP (Q103↑), or GFP (GFP↑) was realized by yeast transformants containing plasmid p426GPD–SWI1, p416GPD–URE2NPDGFP, p426GPD–Q103GFP, or p426GPD–GFP. Log-phase cultures were induced for 48 hr upon addition of 100 μM CuSO₄ (copper) before spreading onto SC–ade plates. Acquired ade⁺ isolates ([PSI⁺] candidates) were quantified for [PSI⁺] de novo formation frequencies after confirming their curability by 5 mM GdnHCl treatment. Shown are results from three independent experiments. (B) Rnq1-CFP signals of the indicated strains with a single copy of RNQ1-CFP integrated at the TRP1 locus, whose expression is driven by the indicated promoters. (C) [SWI⁺] can facilitate [PSI⁺] conversion. Sup35NM-GFP was overproduced with plasmid pCUP1–NMGFP (NMGFP↑). And “vector” represents an empty vector as control. [PSI⁺] induction was performed similarly to that described in A. Cultures were spotted to the indicated plates with a serial dilution and images were taken after 7 days of incubation for SC–ade plates and 3 days for other plates. (D) Sup35NM-GFP exhibits different aggregation patterns in [psi⁻], premature [PSI⁺] (pre[PSI⁺]), and mature [PSI⁺] (m[PSI⁺]) cells during [PSI⁺] prionogenesis facilitated by [SWI⁺]. (E) To quantify Pin⁺ activities, three independent spreading-based [PSI⁺] induction assays were conducted with a methodology similar to that described for experiments shown in A. (F) Nine isogenic pairs of [PIN⁺][SWI⁺] and [PIN⁺][swi⁻] strains and other control strains with the indicated prion backgrounds were compared for their Pin⁺ activities using a spotting assay similar to that used in B. Shown are results of two representative pairs of such strains and controls. Images were taken after 5 days incubation on plates. Left: prion statuses upon transformation of plasmid p416TEF–NQYFP (for Swi1) or pCUP1–RNQ1GFP (for Rnq1).

Figure 2

[SWI⁺] can significantly promote de novo formation of [PIN⁺]. Strains used in this figure all have an integrated TEF–RNQ1-CFP. (A) Top: a [pin⁻][SWI⁺][PSI⁺] isolate (a [PSI⁺] derivative of a [pin⁻][SWI⁺][psi⁻] strain upon overproduction of Sup35NM–YFP but no longer carrying any plasmids) that initially showed diffusible Rnq1-CFP signals gave rise to some rod-like Rnq1-CFP aggregates (pre[PIN⁺]) in a small portion of cells, which eventually became dot-shaped Rnq1-CFP foci in mature [PIN⁺] (m[PIN⁺]) isolates upon colony purification. Bottom: similar conformational changes of Rnq1-CFP and maturation process were observed in [pin⁻][SWI⁺][psi⁻] cells. Shown are representative images. (B) Quantification of spontaneous frequency of [PIN⁺] formation in the indicated strains, which were cultured in liquid YPD for 48 hr before spreading onto YPD plates. Individual colonies were examined for heritable Rnq1-CFP aggregation. Shown are combined results of percentages of examined colonies containing [PIN⁺] as judged by the presence of inheritable Rnq1GFP aggregates. The absolute numbers of [PIN⁺] colonies vs. the total examined are also shown. (C) Stability of [PSI⁺], [PIN⁺] and [SWI⁺] when two or three of them coexist. The indicated strains carrying different prions were grown in YPD medium for 48 hr and retested for their prion status after spreading onto YPD plates. The colony color on YPD plates, Rnq1-CFP signals, and raffinose utilization efficiency were used to score the status of [PSI⁺], [PIN⁺], and [SWI⁺], respectively. Combined results are shown (n, total number of colonies examined)

Figure 3

Aggregates formed spontaneously or induced by a preexisting prion are more prionogenic compared to those formed upon overexpression. (A) Aggregation propensity of Swi1-YFP is depended on its expression levels. (B) Swi1-YFP aggregates acquired in the experiment shown in A were gradually lost upon passaging when Swi1-YFP was constitutively overproduced. (C) Transient overproduction of Swi1 led to aggregation of endogenous Swi1–GFP in a BY4741 SWI1–GFP tagged non-prion strain. (D) Cells harboring Swi1–GFP aggregate shown in C were sorted by flow cytometry. Forward scatter (FSC) height and green fluorescence emission (FL1) intensity are plotted as shown. (E) The nonheritable Swi1–GFP aggregates could not be stabilized upon passage. Hundreds of isolates were assayed and representative results are shown. (F) A TEF1–RNQ1–CFP-integrated [pin⁻] strain was transformed with p426GAL1–SWI1 or an empty vector. Rnq1-CFP aggregates were examined at the indicated time points after galactose addition. (G) Rnq1-CFP aggregates formed upon Swi1 overproduction shown in F could not be successfully transmitted to progeny upon passage. (H and I) Non-TEF1–RNQ1-CFP integrated [PSI⁺][pin⁻] ([PSI⁺]) and [psi⁻][pin⁻] (non-prion)) strains were transformed with plasmid pCUP1–RNQ1GFP. As described in Materials and Methods, Rnq1–GFP aggregates generated after incubation at 4° for 14 days on SC–ura with or without copper salt were assayed for inheritability. (H) Summary of percentages and absolute numbers of isolates harboring heritable Rnq1–GFP aggregate- vs. the total aggregate-containing isolates. (I) Representative result showing heritable and nonheritable aggregates upon passaging.

Figure 4

Preexisting [PIN⁺] aggregates and their interaction with newly produced Sup35NMYFP aggregates during [PSI⁺] prionogenesis. Strains used in these experiments all carried an integrated copy of the TEF1–RNQ1-CFP. Shown are representative fluorescence patterns of YFP, Sup35-NMYFP (NMYFP), and Rnq1-CFP during [PSI⁺] initiation and maturation process for each indicated strain. Initial Rnq1-CFP aggregates in [PIN⁺] cells were dot shaped (top right). Upon NMYFP overproduction (from pRS316CUP1–NMYFP), NMYFP ring/rods appeared only in prion-containing strains (observed after 16 hr overproduction). Note that the ring-/rod-shaped NMYFP signals are significantly overlapped with the remodeled Rnq1-CFP aggregates in cells harboring [PIN⁺]. In mature [PSI⁺] cells, both of the NMYFP and Rnq1-CFP aggregates were mostly dot shaped and essentially not overlapping (bottom). In rare cases, Rnq1–CFP rod-like aggregates could appeared in mature [PSI⁺] cells that were derived from either [pin⁻] or [PIN⁺], and they overlap with Sup35NM–YFP aggregates (bottom, red arrows). Cells that occasionally lost [PIN⁺] or [PSI⁺] aggregates are indicated with white arrows.

Figure 5

Interaction of newly generated premature [PSI⁺] aggregates with preexisting heterologous prion aggregates. The TEF1–RNQ1-CFP-integrated strains with indicated prion backgrounds were transformed with pCUP1–NMYFP and induced in liquid medium as described in the legend of Figure 4. The Sup35NM–YFP ring-/rod-like aggregates and Rnq1–CFP signals were pictured in premature [PSI⁺] cells and merged. Note that in the early stages of [PSI⁺] prionogenesis, there are remarkable overlapping signals between Sup35NM-YFP and Rnq1-CFP in [PIN⁺][swi⁻] strain. Such overlapping signals are significantly reduced in [PIN⁺][SWI⁺] cells and essential absent in [pin⁻][SWI⁺] cells.

Figure 6

Preexisting [SWI⁺] aggregates and their interaction with newly formed Sup35NM-CFP and Rnq1-CFP aggregates during [PSI⁺] and [PIN⁺] prionogenesis and maturation. (A) Yeast strains containing the indicated prion(s) were transformed with plasmids pRS316CUP1–NMCFP and p413TEF–NQYFP and induced for [PSI⁺] formation. Images were taken 16 hr after addition of CuSO₄ when ring/rod-shaped Sup35NM-CFP (MNCFP) aggregates were formed in premature [PSI⁺] cells (pre[PSI⁺]) cells. The ring-/rod-shaped NMCFP signals are significantly overlapped with the remodeled Swi1NQ-YFP (NQYFP) aggregates in cells harboring [SWI⁺] (left). Mature [PSI⁺] (m[PSI⁺]) isolates were reexamined for NMCFP and NQYFP fluorescence patterns, which were mostly dot shaped and essentially not overlapping. Rare overlapping signals are indicated. See Figure S4 for more images. (B) An RNQ1-CFP-integrated [SWI⁺][pin⁻] strain was transformed with the plasmid p413TEF–NQYFP and grown in SC–his liquid medium for 2 days (∼40 hr) and screened for the rod-like premature Rnq1-CFP (pre[PIN⁺]) aggregates; NQYFP signals were also recorded. Upon colony purification, stably inherited aggregates of Rnq1-CFP and NQYFP were examined in mature [PIN⁺] (m[PIN⁺]) cells (right).