Introducing new [PSI +] variants

All new [PSI+] variants reported in the present work are summarized here for easy reference. By transient Sup35 overexpression, 6 new, rare variants were induced de novo in T160M cells or in cells containing ssb1/2∆, ssz1∆, and upf1∆ mutations (Table 1; Fig. 1). Cells propagating the variants were streaked on agar plates for several times, and single colonies were checked each time to ensure variant-type robustness (see Materials and Methods for the variant-typing protocol). The 6 variants were further transferred to wild-type and T160M cells by cytoduction (i.e. cytoplasm mixing without exchange of nuclei, see Materials and Methods) and re-checked. All of them were able to propagate in at least one of the wild-type or T160M cells without the deletions (Table 2).

Table 1 The host range of the new [PSI+] variantsFig. 1

New [PSI+] variants. (a) [PSI+] variants (labeled on the left) are distinguished by colony colors. Cells are crossed with prion-less testers transformed with plasmids expressing full-length Sup35 or single mutations (top). The diploid colonies show variant-specific color patterns. Ze, Hdp, and B7 are crossed with wild-type testers; V7, V8, and V9 are with T160M testers. (b) The variants are distinguished by GFP labeling. Cells are crossed with testers expressing Sup(1–61)-GFP, its single mutants, or Sup(1–40)-GFP (indicated on the top). (+) indicates particulate GFP labeling, as shown in panel (d), right; (–) indicates diffused GFP labeling as in (d), left. Ze, Hdp, and B7 are crossed with wild-type testers; V7, V8, and V9 are with T160M testers. (c) and (d) Ze is a proper [PSI+] variant although it is unresponsive in variant typing. Ze is cured by 3 mM guanidine hydrochloride (c) and 100% labeled by Sup(1–80)-GFP (d)

Table 2 Cytoduction of the new variants. The recipient cell type is shown on top[PSI +] induction in yeast cells with ssb1/2∆, ssz1∆, or upf1∆ mutation

[PSI+] variants induced in yeast strains with ssb1/2∆, ssz1∆, or upf1∆ mutation were selected by nonsense readthrough of the ade1-14 allele, which encodes a premature UGA stop codon. Two independent experiments were performed for each strain. In first experiments, [PSI+] colonies were selected by its pink or white color on rich media. (Yeast carrying the ade1-14 mutation is red on rich media. [PSI+] suppresses the nonsense mutation and reduces the redness.) In second experiments, [PSI+] colonies were directly obtained on synthetic media lacking adenine without reference to colony color (cells lacking the prion could not grow). The majority of [PSI+] isolates turned out to be VH, VK, and VL (Table 3). They are well-studied [PSI+] variants frequently obtained in wild-type strains. Two new variants did appear. [Ze] and [Hdp] were induced in ssb1/2∆ and ssz1∆ strains, respectively (Table 3; also see Fig. 1; Table 1). They however could propagate in wild-type cells with normal Ssb1/2 and Ssz1 activities (Table 2). No variant curable by Ssb1/2, Ssz1, or Upf1 was detected.

Table 3 De Novo [PSI+] induction

The above conclusion is supported by an additional observation. [PSI+] colonies were mated with wild-type testers carrying a set of reporter plasmids to determine the variant type. All but one colony continued propagating the prion in the diploids where Ssb1/2, Ssz1, and Upf1 functions were restored by complementation. The single exception was a white colony with SSZ1 deletion, which became red when forming diploids. The colony was transformed directly with plasmids expressing Sup(1–61)-GFP (containing the first 61 amino-acid residues of Sup35 fused with the green fluorescent protein), Sup(1–80)-GFP, and Sup(1-114)-GFP to label possible [PSI+] particles in vivo. None of the cells transformed with Sup(1–61)-GFP contained labeled particles. About 30% of the cells transformed with Sup(1–81) or Sup(1-114) had GFP puncta, but the occurrence rate was too low for a [PSI+] cell (near 100%). Moreover, the same 30% rate was observed in control experiments where the original deletion strain used for [PSI+] induction ([psi−] [PIN+] ∆ssz1) was similarly treated. The puncta were thus considered as an artifact, inadvertently induced by the GFP fusion proteins in a [PIN+] background.

To investigate further, the cytoplasm of the white colony was transferred to prion-free recipients that had the same ssz1∆ mutation. All resulting cytoductants formed red colonies (30/30). Clearly, the whiteness was not due to [PSI+]; it was most likely caused by a recessive mutation on the yeast chromosome.

[PSI +] induction in a Hsp104T160Mssz1∆ upf1∆ strain

[PSI+] induction was next performed with T160M cells with ssz1∆ and upf1∆ mutations. Experiments were executed as described above except that for variant typing, [PSI+] colonies selected on adenine-less media were mated with both wild-type and T160M tester cells. The extra work ensured more accurate variant type assignment. Variants that were stable in T160M cells but mutated in wild-type cells (or Hsp104WT/Hsp104T160M heterozygotes) could be confidently identified. The experimental result is listed in Table 3.

The variants induced here could be grouped into 3 classes, based on compatibilities with wild-type and T160M cells in the absence of the deletions.

Class (I): variants that only propagate with Hsp104WT. Surprisingly, VK and VL variants that normally cannot propagate with Hsp104T160M showed up here (Huang et al. 2020). They were unambiguously typed when colonies were crossed with wild-type testers (i.e. in Hsp104WT/Hsp104T160M heterozygotes) but were cured with T160M testers (Hsp104T160M homozygotes), exactly as expected. In addition, a new variant, B7, was isolated (Fig. 1). It could infrequently change to VK in wild-type cells. When transferred to T160M cells, it mutated to the B6 variant (Huang and King 2020) and disappeared (Tables 1 and 2).

Class (II): variants that only propagate with Hsp104T160M. There were B6, V2, V6 and a new variant, named V7 (see Fig. 1; Tables 1 and 2). They were cured in wild-type cells, but could escape total elimination by changing to VH or VK.

Class (III): variants that can propagate in both wild-type and T160M cells. VH was the most abundant (VH is however less stable in T160M cells, lost in mitosis at a low rate). The UnS variant was also identified, which propagates faithfully with Hsp104T160M but can change to VH and VK in wild-type cells (Huang et al. 2020; King 2022).

In conclusion, double deletion of SSZ1 and UPF1 in T160M cells appears to generate an accommodating environment for all [PSI+] variants, especially those of Class I, which otherwise can only be maintained with wild-type Hsp104.

The ssz1∆ upf1∆ double mutation moderately enhances [PSI +] induction in Hsp104T160M cells

The frequency of de novo [PSI+] induction was estimated in T160M cells with and without the ssz1∆ upf1∆ double mutation. The full-length Sup35 protein was overexpressed from a multi-copy plasmid in the cell. Cultures were streak on rich media. About 120 colonies were randomly picked and gridded on agar plates. They were forced to lose the plasmid and then assayed for [PSI+] status by colony color. Nearly half of the T160M colonies and 90% of the T160M ssz1∆ upf1∆ colonies propagated [PSI+] (54/120, 70/119 and 103/120, 107/120, respectively). The frequency of the former was consistent with past observations, and was close to the value obtained in the first paper reporting [PSI+] induction by Sup35 (in wild-type strains; Chernoff et al. 1993).

Table 3 shows that 57–62% of the [PSI+] colonies induced in T160M ssz1∆ upf1∆ cells were VH colonies, and 37 − 30% were VK or VL colonies. With a 90% overall [PSI+] induction rate measured here, there would be about 51–60% of the colonies that propagated VH (57–62% times 90%), 33 to 27% colonies for VK or VL and 10% [psi−] colonies. On the other hand, [PSI+] induction in T160M cells generates mostly the VH variant (> 90%; Class II variants are also induced, but infrequently; Huang et al. 2020), so there would be roughly 50% VH colonies and 50% [psi−] colonies for T160M cells. It seems that the increase in [PSI+] induction with the ssz∆1 upf1∆ mutation could be largely attributed to the appearance of VK and VL (Class I variants).

[PSI +] variants induced in 74-D694 are not cured by Ssz1 or Upf1 overexpression

Son and Wickner (2018, 2020, 2022) used a different setting to induce and select [PSI+] variants. The prion was induced by overexpression of Sup35(1-253) with a galactose-inducible Gal1 promoter and selected with the ura3-14 marker (Monogaran et al. 2006). The inducer and selection marker were conveniently carried by a single centromere-based plasmid p1520 (Wickner et al. 2017). Using this method, [PSI+] variants were induced again in the 74-D694 background with ssz1∆, upf1∆, and Hsp104T160Mssz1∆ upf1∆ mutations. One hundred and twenty Ura+ colonies were randomly selected for each mutant. Figure 2 shows that none of the colonies were cured of [PSI+] when mated with wild-type cells overexpressing Ssz1 or Upf1.

Fig. 2

p1520-induced [PSI+] variants are not cured by Ssz1 or Upf1 overexpression in the 74-D694 background. (a) [PSI+] variants induced in upf1∆ cells are not cured by Upf1 overexpression. Randomly picked Ura+ isolates (left) are crossed with wild-type cells overexpress Upf1 (right). No red diploid appears. All isolates on the master plate are variant-typed and thus confirmed to be [PSI+] to begin with. (b) [PSI+] variants induced in ssz1∆ cells are not cured by Ssz1 overexpression. Nonsense readthrough is elevated in ssz1∆ cells. The elevation is corrected in diploids. (c) [PSI+] variants induced in Hsp104T160Mupf1∆ ssz1∆ cells are not cured by Ssz1 or Upf1 overexpression. There are 21 Kbar variants (4 random examples are circled), which are cured in T160M homozygotes but not in WT/T160M heterozygotes. Kbar is a Class I variant (see text for definition; King 2022), which can propagate with Hsp104WT but not Hsp104T160M unless SSZ1 and UPF1 are deleted. The colony in rectangle propagates B3, which is a Class II variant (i.e. compatible with Hsp104T160M but not Hsp104WT (King 2022). It however can survive in WT/T160M heterozygotes

The frequencies of [PSI+] induction in the 74-D694 strain, reported above, were significantly higher than that observed by Son and Wickner in the BY4742 strain. It can be argued that multiple rounds of induction have occurred in a single 74-D694 cell, and Ssz1-, Upf1-, and Ssb1/2-curable variants were competed out by a co-induced variant, therefore the failure to isolate them. To rule out this possibility, strains carrying p1520 were grown in glucose media, which suppressed the expression of Sup35(1-253), turning off [PSI+] induction. Spontaneously occurred Ura+ colonies were then selected. Small colonies were preferentially picked with the help of a dissection microscope to maximize the isolation of weak [PSI+] variants (but at the expense of getting false positives, especially given that the background nonsense suppression is elevated in the deletion strains, and there were more than one copy of plasmid-carried ura3-14 markers in the cell, enhancing the chance of nonsense suppression). No novel variants were uncovered (Table 4). And again, none of the isolated [PSI+] colonies lost the prion when mated with wild-type cells overexpressing Ssz1 or Upf1 (not shown).

Table 4 The variant type of spontaneously formed ura++ coloniesThe ssz1∆ upf1∆ double mutation does not help Hsp104WT to support incompatible variants

I next asked if deleting SSZ1 and UPF1 could also enable wild-type cells to propagate Class II variants that are not supported otherwise. Class II Variants V6 and V7 were transferred to recipient cells expressing wild-type Hsp104 and carrying the deletions (Hsp104WTssz1∆ upf1∆). The donor variants were lost in the cytoductants (Table 5). Instead, there were several cell colonies propagating VH, consistent with the fact that V6 can mutate in wild-type cells (King 2022). The result indicated a negative answer.

Table 5 Cytoduction of class II variants and VK. The recipient cell type is shown on top

The appearance of “contaminating” VH colonies in the experiment was not ideal, but besides V6 and V7, other known Class II variants were also mutable. I therefore tried isolating new variants that rarely mutated and only propagated with Hsp104T160M. Variants V8 and V9 were obtained after some efforts (see Fig. 1; Tables 1 and 2). They were duly transferred into the Hsp104WTssz1∆ upf1∆ recipient. All of the resulting cytoductants lost [PSI+] (Table 5). There was an additional consideration. Kiktev et al. (2015) showed that deletion of SSZ1 causes cytosolic release of ribosome-associated Ssb1/2, and that enhances [PSI+] curing. To remove this effect, SSB1 and SSB2 genes were further deleted from the recipient strain. V8 and V9 still could not propagate (Table 5). As a control, the Class I variant VK was introduced into the quadruple mutant and was observed to stably propagate, as it should (Table 5). Therefore, knocking out SSZ1 and UPF1 in a wild-type cell did not make it hospitable for Class II variants.

I next performed cytoduction experiments with B3, which is a Class II variant but can propagate in wild-type cells if the SUP35 gene is carried by a centromere-based plasmid instead of by the chromosome (i.e. sup35∆ [YCp111-Sup35]), indicating that just a tad higher Sup35 expression is able to rescue B3 propagation with Hsp104WT (Huang and King 2020; King 2022). Similar to other Class II variants, B3 could not propagate in the quadruple mutant in the absence of episomal SUP35 (Table 5). The implication of this finding is discussed below.

Effects of a single gene deletion on [PSI +] propagation

To gauge individual contributions of ssz1∆ and upf1∆ for prion propagation, Class I variants VK, VL and B2 (Huang and King 2020) were transferred to Hsp104T160Mssz1∆ and Hsp104T160Mupf1∆ strains by cytoduction. [PSI+] particles in the cytoductants were detected by labeling with Sup35-GFP fusion proteins (see Materials and Methods for details). For control, the variants were first transferred to T160M recipients without the deletions. As expected, they were unable to propagate (Fig. 3a). Single upf1∆ mutation only improved VL propagation. In contrast, ssz1∆ allowed most cytoductants to propagate [PSI+], although some cells in the colonies still lost prion in mitosis, as judged by the absence of labeled particles. The loss was largely mitigated in ssz1∆ upf1∆ cytoductants, thus revealing the subtle contribution of upf1∆ for all variants (Fig. 3a).

Fig. 3

ssz1∆ and upf1∆ rescue the propagation of [PSI+] and [PIN+]. (a) [PSI+] variants incompatible with Hsp104T160M (indicated on the left) are introduced into hosts with additional ssz1∆ and upf1∆ mutations (labeled on top). The resulting colonies are checked for [PSI+] maintenance. More than 1000 cells of each colony are observed for prion particles by GFP labeling. The proportion of cells containing prion particles are estimated. Proportions are coded by color and by location such that a higher value is represented by a lighter color and a lower position in the entry. For example, regarding B2 propagating in T160M ssz1∆ upf1∆ (right bottom), among the 6 colonies checked, there is one containing no prion (darkest gray, uppermost position), 3 having 50–90% cells with prion particles (3/6; second lightest, lower middle), and 2 colonies that are 100% labeled (lightest, lowermost). (b) [PIN+] propagation in T160M cells. (c) [PSI+] propagation in ∆N cells. (d) [PIN+] propagation in ∆N cells

Deleting SSZ1 and UPF1 also improves the propagation of [PIN +] variants

I next tested if deleting SSZ1 or UPF1 could similarly help other yeast prion to propagate with deficient Hsp104. Two [PIN+] variants, [Very High] and [Medium], which propagate well in wild-type cells but poorly with Hsp104 mutants, were used for the experiment (Bradley et al. 2002; Huang et al. 2020). Wild-type strains harboring the variants were crossed with T160M ssz1∆ upf1∆ cells and the resulting diploids were induced to undergo meiosis. Haploid progenies were genotyped and checked for [PIN+] by RNQ1-GFP labeling ([PIN+] is the prion form of Rnq1). The two [PIN+] variants could still propagate in T160M cells, but exhibit some mitotic loss (Fig. 3b). The mitotic instability could be somewhat relieved with additional ssz1∆ or upf1∆ mutation. For [Very High], either mutation resulted in complete relief. For [Medium], double deletion was needed to achieve total rescue. The results indicate that ssz1∆ and upf1∆ could also help T160M cells to maintain the [PIN+] variants.

The residual propagation of the [PIN+] variants in T160M cells can be largely avoided in cells expressing Hsp104∆N, lacking residues 2- 147 (designated ∆N hereinafter; Huang et al. 2020). The ∆N mutant, like T160M, also exhibits weaker activities, fails to support VK and VL variants, but can nevertheless sustain the propagation of Class II [PSI+] variants. Experiments were performed again by crossing the wild-type yeast strains with ∆N ssz1∆ upf1∆ cells, followed with meiosis and spore analysis.

Deletion of UPF1 allowed ∆N cells to partially support the [PIN+] variants (Fig. 3d). In contrast, none of the ∆N cells with ssz1∆ had prion particles. However, ssz1∆ might still have some positive influence since the ssz1∆ upf1∆ double mutation supported [PIN+] inheritance much better than upf1∆ alone. As an internal control, all haploid cells inheriting wild-type HSP104 (instead of the ∆N allele) propagated [PIN+] with 100% GFP-labeling regardless of the deletions. Thus, deleting UPF1 and SSZ1 could help [PIN+] propagation in ∆N cells.

[PSI +] propagation in Hsp104∆N backgrounds

To compare [PSI+] and [PIN+] on a more equal footing, VK, VL and B2 were checked for propagation in ∆N cells, using cytoduction for prion transfer. Indeed, [PSI+] propagation was rescued by ssz1∆ and upf1∆ mutations. Like the [PIN+] variants, VK, VL, and B2 propagated better with upf1∆ than with ssz1∆, and the double deletion supported near-perfect propagation (Fig. 3c). The striking similarity between [PSI+] and [PIN+] reaffirms that general mechanisms are involved in the improvement of prion propagation.

Deletion of UPF1 and SSZ1 results in higher Hsp104∆N expression

Western analysis was performed with cell extracts. The expression level of Hsp104∆N correlated nicely with the extent of improvement observed for prion propagation (Figs. 3c and d and 4b). Cells with SSZ1 deletion showed higher Hsp104∆N expression than the control; the expression in upf1∆ cells was even higher. And the double deletion resulted in the highest protein level (Fig. 4b). In contrast, Hsp104T160M expression levels were similar across all type of cells. The expressions of Ssa1/2, Sis1, and Sup35 were also analyzed by Western blots; they did not differ significantly among different cell types (not shown).

Fig. 4

Hsp104 expression. (a) An upstream out-of-frame ATG codon (red rectangle) was fortuitously generated when constructing Hsp104∆N. The BamHI restriction site is in italics. (b) Hsp104 expression in different cell types (indicated on the top) is analyzed by Western blot. ∆N: Hsp104∆(2–147). T160M: Hsp104T160M. Loading control: glucose-6-phosphate dehydrogenase (G-6-PDH; on bottom half of the gel). Marker sizes: 100 and 55 KDa. The arrow indicates degradation artefact

A careful comparison of Fig. 3a and c reveals that upf1∆ had a more pronounced effect in ∆N cells than in T160M cells. This is not unexpected. In constructing ∆N strains (but not T160M strains), a restriction site was introduced between the promoter and the coding sequence of Hsp104∆N, which inadvertently created an upstream, out-of-frame ATG triplet in the transcript (Fig. 4a). Translation initiated from the fortuitous start would terminate prematurely, making the transcript an NMD target. Knocking out UPF1 stabilized the transcript. The resulting higher expression of Hsp104∆N could compensate for the weak disaggregase activity and help the [PSI+] variants to propagate. Quantitative reverse transcription polymerase chain reaction (RT-qPCR) confirmed that upf1∆ cells contained about 1.5 times more Hsp104∆N transcripts than the Upf1+ counterpart (two sets of experiments were performed, using [psi−][pin−] and [psi−][PIN+] strains. The expression ratios were 1.40 ± 0.18 and 1.618 ± 0.31, respectively).

Propagation properties of the D3 variant

Experiments with the D3 variant were informative for understanding the pro-prion effect of the Hsp104T160Mssz1∆ upf1∆ mutation. There are 4 [PSI+] variants, A2, A3, B1, and D3, which can barely propagate in wild-type cells, T160M cells, or cells overexpressing the wild-type Hsp104, but can be maintained in wild-type cells as long as the SUP35 gene is relocated from the chromosome to a low-copy-number plasmid (Huang and King 2020; King 2022). The 4 variants were cytoduced to Hsp104T160Mssz1∆ upf1∆ cells; only D3 survived. An interesting situation now arises: in cells with normal Sup35 expression, neither high nor low disaggregase activities afforded by the wild-type Hsp104 and the T160M mutant, respectively, sufficiently support D3. However, the triple mutant propagated the variant. Further experiments showed that D3 was not cured by Ssz1 or Upf1 overexpression (Fig. 5), and even without extra Sup35, the variant could be maintained in Hsp104T160MSsz1+Upf1+ cells that co-expressed Hsp104WT (Table 6). Hsp104 is a hexamer. A mixture of hetero-hexamers was generated in the cells. The resulting intermediate activities turned out just right for D3 (see below for more discussion).

Fig. 5

Ssz1 or Upf1 overexpression does not perturb the D3 variant of [PSI+]. D3 can be maintained in both wild-type cells expressing Sup35 from a plasmid and in Hsp104T160Mssz1∆ upf1∆ mutants with normal Sup35 expression (labeled on the top). The cells are crossed with wild-type or T160M testers overexpresses Ssz1 or Upf1 from a multi-copy plasmid (labeled on the left). No destabilization is observed as colony colors stay the same with or without Ssz1/Upf1 overexpression. Also note that D3 propagates better in Hsp104WT/T160M heterozygotes (lighter color) than in homozygotes. WT: Hsp104WT. T160M: Hsp104T160M. [YEp195]: empty plasmid control

Table 6 Cytoduction of D3