A heritable switch in carbon source utilization driven by an unusual yeast prion

Abstract

Several well-characterized fungal proteins act as prions, proteins capable of multiple conformations, each with different activities, at least one of which is self-propagating. Through such self-propagating changes in function, yeast prions act as protein-based elements of phenotypic inheritance. We report a prion that makes cells resistant to the glucose-associated repression of alternative carbon sources, [GAR(+)] (for "resistant to glucose-associated repression," with capital letters indicating dominance and brackets indicating its non-Mendelian character). [GAR(+)] appears spontaneously at a high rate and is transmissible by non-Mendelian, cytoplasmic inheritance. Several lines of evidence suggest that the prion state involves a complex between a small fraction of the cellular complement of Pma1, the major plasma membrane proton pump, and Std1, a much lower-abundance protein that participates in glucose signaling. The Pma1 proteins from closely related Saccharomyces species are also associated with the appearance of [GAR(+)]. This allowed us to confirm the relationship between Pma1, Std1, and [GAR(+)] by establishing that these proteins can create a transmission barrier for prion propagation and induction in Saccharomyces cerevisiae. The fact that yeast cells employ a prion-based mechanism for heritably switching between distinct carbon source utilization strategies, and employ the plasma membrane proton pump to do so, expands the biological framework in which self-propagating protein-based elements of inheritance operate.

Figure 1.

[GAR⁺] shares the genetic characteristics of yeast prions. (A) Mating of [gar⁻] MATa to [GAR⁺] MATα in the W303 background. Resultant diploids show semidominant [GAR⁺] with a mixed population of large colonies (“strong”) and small colonies (“weak”). All spot tests shown are fivefold dilutions. Diploids are selected prior to plating to ensure that they are a pure population. (B) Tetrad spores from the “strong” [GAR⁺]. Diploids in A show non-Mendelian segregation of [GAR⁺]. (C). Cytoduction shows cytoplasmic inheritance of [GAR⁺]. The [GAR⁺] donor is 10B URA3⁺ his3⁻ ρ⁺ kar1-1 and the acceptor is W303 ura3⁻ HIS3⁺ ρ⁰ KAR1. The [GAR⁺] donor is therefore capable of growing on glycerol but the [gar⁻] acceptor is not; “mixed” cells were selected for growth on glycerol ([GAR⁺] cytoplasm) and SD-his 5-FOA ([gar⁻] nucleus and counterselection against the [GAR⁺] nucleus). (D). [GAR⁺] frequency in various laboratory strains. Data are shown as mean ± standard deviation (n = 6). (E). Tetrad spores from a [GAR⁺] diploid with the genotype hsp104∷LEU2/HSP104. Δhsp104 spores are still [GAR⁺]. (F). Tetrad spores from a [GAR⁺] diploid with the genotype ssa1∷HIS3/SSA1 ssa2∷LEU2/SSA2. Δssa1Δssa2 spores are no longer [GAR⁺].

Figure 2.

The Snf3/Rgt2 glucose signaling pathway affects [GAR⁺]. (A) Hxt3-GFP signal in [gar⁻] and [GAR⁺] cells (S288c background) by fluorescence microscopy. (B) Frequency of [GAR⁺] in knockouts of members of the Snf3/Rgt2 glucose signaling pathway. Δsnf3 is completely resistant to glucosamine, and therefore [GAR⁺] frequency could not be measured. Furthermore, the frequency of spontaneous glucosamine-resistant colonies in the Δrgt1, Δstd1, and Δmths1 strains was close to the rate of genetic mutation, and therefore these colonies might not carry the actual [GAR⁺] element. Overall, this pathway is enriched for genes that alter [GAR⁺] frequency when knocked out relative to the library of nonessential genes (P = 8 × 10⁻⁶, Fisher's exact test). (C) The Snf3/Rgt2 glucose signaling pathway. (Adapted with permission from Moriya and Johnston 2004; ©2004 National Academy of Sciences, USA.) (D) Measurement of [GAR⁺] frequency following overexpression of Snf3/Rgt2 pathway members. Data are shown as mean ± standard deviation (n = 6). STD1 strongly induces conversion to [GAR⁺] and MTH1 blocks it. (E, top) Tetrad spores from a [GAR⁺] diploid with the genotype std1∷kanMX/STD1. (Bottom) Spores from top crossed to a [gar⁻] strain with a wild-type STD1 allele.

Figure 3.

Pma1 is involved in [GAR⁺]. (A) Native gel of Pma1, Std1, and Mth1 in [gar⁻] and [GAR⁺]. Either Std1 (left) or Mth1 (right) was tagged with six tandem HA tags and samples were processed as described below from [gar⁻] and [GAR⁺] strains of each background. (Bottom right) Total, supernatant (sup.), digitonin soluble (det. sol.), and digitonin-insoluble (insol.) fractions were run on SDS gels and probed for Pma1 and Std1 or Mth1 as a fractionation control. No differences in Pma1, Std1, or Mth1 levels or localization were detected between [gar⁻] and [GAR⁺]. (Top right) Blots of the total fraction were stained with Ponceau Red to confirm equal amounts of starting material. (B) Measurement of [GAR⁺] frequency in knockout mutants of genes previously shown to affect (Δsur4, Δlst1) (Roberg et al. 1999; Eisenkolb et al. 2002) or not affect (Δlcb3, Δlcb4, Δdpl1, Δatg19) (Gaigg et al. 2005; Mazon et al. 2007) attributes of wild-type Pma1. Graph represents the mean ± standard deviation (n = 6). (C) Mutants in phosphorylation sites at the C terminus of Pma1 affect [GAR⁺] frequency. Starting strain is haploid, [gar⁻], genotype pma1∷kanMX with p316-PMA1. p314-PMA1 carrying wild-type PMA1 or mutants of interest were transformed into the starting strain and then p316-PMA1 plasmid selected against by growth on 5-FOA. Graph represents the mean ± standard deviation (n = 6). P-values are the binomial distribution of the mean. (D) Pma1 mutants that increase [GAR⁺] frequency show decreased levels of Hxt3-GFP. Graph represents the mean ± standard deviation (n ≥ 6) and P-values were determined using the χ² test. Strain background is a hybrid of W303 and S288C.

Figure 4.

Alterations to Pma1 affect [GAR⁺]. (A) [GAR⁺] induction by transient overexpression of PMA1 in a wild-type background. Data are shown as the mean of [GAR⁺] frequency ± standard deviation (n = 6). Western is total protein probed with αPma1 antibody and quantified using Scion Image. (Right) The blot was stained with Ponceau Red to confirm equal loading. (B) Propagation of [GAR⁺] is impaired in PMA1Δ40N Δstd1 double mutants. Tetrad spores from a [GAR⁺] diploid with the genotype GAL-PMA1Δ40N/PMA1 std1∷kanMX/STD1 were crossed to a [gar⁻] strain with wild-type PMA1 and STD1 alleles. PMA1Δ40N Δstd1 spores cannot propagate [GAR⁺] to wild-type [gar⁻] yeast. The few glucosamine-resistant colonies found in the PMA1Δ40N Δstd1 background exhibit standard, Mendelian inheritance of the glucosamine resistance phenotype and thus do not carry the [GAR⁺] element.

Figure 5.

[GAR⁺] exhibits a Pma1-dependent species barrier. (A) [GAR⁺] frequency of S. bayanus and S. paradoxus cells grown at 30°C (left), the optimal growth temperature of S. paradoxus, or 23°C (right), the optimal growth temperature of S. bayanus. Data are shown as the mean of [GAR⁺] frequency ± standard deviation (n = 6). (B) Substitution of PMA1 from S. cerevisiae with PMA1 from S. bayanus or S. paradoxus prevents [GAR⁺] propagation. Starting strain is haploid, [GAR⁺], genotype pma1∷kanMX with p316-PMA1 S. cerevisiae as a covering plasmid. p314-PMA1 carrying PMA1 from S. cerevisiae (S.c., top), S. paradoxus (S.par., middle), or S. bayanus (S.bay., bottom) was transformed into the starting strain and p316-PMA1 S.c. selected against by replica plating to 5-FOA (S.c. 1N, S.p. 1N, or S.b. 1N). These haploids were mated to a wild-type S. cerevisiae [gar⁻] background, restreaked twice, and tested for [GAR⁺]. Representative data from three independent experiments are shown.

Figure 6.

Pma1 and the Rgt2/Snf3 glucose signaling pathway We propose that Pma1 acts as a part of the Rgt2/Snf3 signaling pathway. (A) In [gar⁻] glucose-grown cells, Pma1 associates with Mth1. The glucose signal is propagated through Snf3 and Rgt2 to Yck1 and Yck2, which phosphorylate Mth1 and Std1. This phosphorylation marks Mth1 and Std1 for degredation, leaving their interacting partner, Rgt1, free in the cytosol, where it does not repress transcription at the HXT3 locus. (B) Under [GAR⁺] conditions, HXT3 transcription is repressed, which resembles that of cells grown in a carbon source other than glucose. Pma1 associates with Std1, which somehow facilitates the repression of HXT3, possibly by altering the affinity of Std1 for Rgt1. Association with Std1 has been shown previously to facilitate the binding of Rgt1 to DNA (Lakshmanan et al. 2003). The association between Pma1 can either be transient or stable, but either way it aids in the establishment of an altered signaling pathway. This altered pathway is then maintained either by the contained association between Std1 and Pma1 or by a feedback loop within the signaling cascade itself.