Interaction of Prions Causes Heritable Traits in Saccharomyces cerevisiae

Abstract

The concept of "protein-based inheritance" defines prions as epigenetic determinants that cause several heritable traits in eukaryotic microorganisms, such as Saccharomyces cerevisiae and Podospora anserina. Previously, we discovered a non-chromosomal factor, [NSI+], which possesses the main features of yeast prions, including cytoplasmic infectivity, reversible curability, dominance, and non-Mendelian inheritance in meiosis. This factor causes omnipotent suppression of nonsense mutations in strains of S. cerevisiae bearing a deleted or modified Sup35 N-terminal domain. In this work, we identified protein determinants of [NSI+] using an original method of proteomic screening for prions. The suppression of nonsense mutations in [NSI+] strains is determined by the interaction between [SWI+] and [PIN+] prions. Using genetic and biochemical methods, we showed that [SWI+] is the key determinant of this nonsense suppression, whereas [PIN+] does not cause nonsense suppression by itself but strongly enhances the effect of [SWI+]. We demonstrated that interaction of [SWI+] and [PIN+] causes inactivation of SUP45 gene that leads to nonsense suppression. Our data show that prion interactions may cause heritable traits in Saccharomyces cerevisiae.

Fig 1. 2D-DIGE image of proteins forming SDS-insoluble aggregates isolated from the 1-1-D931 [NSI⁺] and 1-1-1-D931 [nsi^-] strains.

Spots corresponding to proteins from 1-1-D931 [NSI⁺] cells are pseudocolored in red (Cy5), while proteins from the 1-1-1-D931 [nsi^-] are pseudocolored in green (Cy3). Yellow spots correspond to proteins present in both samples. A strip with a pH gradient of 5–8 was used. Proteins identified by mass-spectrometry are indicated. The mass spectra of identified proteins are listed in S1–S3 Figs.

Fig 2. [NSI⁺] strain contains [PIN⁺] prion which acts as the enhancer of the nonsense suppression.

(A) SDD-AGE of protein lysates extracted from the 1-1-D931 [NSI⁺] and 1-1-1-D931 [nsi^-] strains expressing pCUP1-RNQ1-CFP(LEU2) plasmid. Protein lysates were treated with 1% SDS at room temperature. SDS-resistant aggregates of Rnq1-CFP were detected using monoclonal rabbit primary antibodies against GFP [E385] (ab32146) (Abcam, Great Britain) and ECL Prime Western Blotting Detection Reagent kit (GE Healthcare, USA). (B) The effects of RNQ1 deletion on the [NSI⁺] phenotypic manifestation. RNQ1 deletion was obtained as described in Materials and Methods. The [nsi^-] and [nsi^-] rnqΔ strains were obtained from the corresponding [NSI⁺] strains by GuHCl treatment. To express RNQ1, the 5-1-1-D931 [NSI⁺] rnqΔ strain and its [nsi^-] derivative were transformed with the YGPM25a02 plasmid containing a genomic fragment encoding RNQ1 under the control of its endogenous promoter. Other strains presented in this Figure were transformed with an empty vector expressing only the LEU2 gene. Transformants were selected on–Leu medium with 150 μM CuSO₄ and replica-plated on–Leu–Ade medium or–Leu medium with 150 μM CuSO₄ containing galactose as the sole carbon source. Images were taken after 5 days of incubation of–Ade plates or after 3 passages on Gal plates.

Fig 3. [SWI⁺] prion is a key determinant of nonsense suppression in [NSI⁺] strains.

(A) Sedimentation analysis of Swi1(1–297)-YFP protein from the 4-1-1-D931 [NSI⁺] and 1-4-1-1-D931 [nsi^-] strains expressing pCUP1-SWI1(1–297)-YFP (URA3) plasmid. Soluble (S) and insoluble (I) fractions were obtained as indicated in Materials and Methods. Swi1(1–297)-YFP was detected using monoclonal rabbit primary antibodies against GFP [E385] (ab32146) (Abcam, Great Britain) and ECL Prime Western Blotting Detection Reagent kit (GE Healthcare, USA). Next, SDD-AGE analysis of insoluble fractions of [NSI⁺] and [nsi^-] strains comprising Swi1(1–297)-YFP was performed. (B) The effects of SWI1 deletion on the [NSI⁺] phenotypic manifestation. SWI1 deletion was obtained as described in Materials and Methods. To express SWI1, the 11-1-1-D931 [NSI⁺] swi1Δ strain was transformed with the YGPM19p21 plasmid from the YSC4613 genomic library, containing a genomic fragment encoding SWI1 under the control of its endogenous promoter. Other strains presented in this Figure were transformed with an empty vector expressing only the LEU2 gene. Transformants were selected on–Leu medium with 150 μM CuSO₄ and replica-plated on–Leu–Ade or–Leu Gal media with 150 μM CuSO₄. Images were taken after 5 days of incubation of–Ade plates or after 3 passages on Gal plates.

Fig 4. Mit1 is not a determinant of the [NSI⁺] factor.

(A) SDD-AGE assay of protein lysates extracted from the 4-1-1-D931 [NSI⁺] and 1-4-1-1-D931 [nsi^-] strains expressing pMIT1-MIT1-GFP(URA3) plasmid. Cells were grown for 48 h at 30°C in liquid–Ura selective medium containing 150 μM CuSO₄. Protein lysates were treated with 1% SDS at room temperature. Mit1-GFP was detected with monoclonal rabbit primary antibodies against GFP [E385] (ab32146) (Abcam, Great Britain) and ECL Prime Western Blotting Detection Reagent kit (GE Healthcare, USA). (B) MIT1 deletion does not affect the [NSI⁺] phenotypic manifestation. MIT1 deletion was obtained as described in Materials and Methods. The [nsi^-] derivative of the 2–936 [NSI⁺] mit1Δ was obtained by GuHCl treatment. To express MIT1, 2–936 [NSI⁺] mit1Δ strain was transformed with YGPM21o12 plasmid from the YSC4613 genomic library containing a genomic fragment encoding MIT1 under the control of its endogenous promoter. Other strains presented in this Figure were transformed with a vector expressing only the LEU2 gene. Transformants were selected on–Leu medium with 150 μM CuSO₄ and replica-plated on–Leu–Ade or–Leu Gal media with 150 μM CuSO₄. Images were taken after 5 days of incubation of–Ade plates or after 3 passages on Gal plates.

Fig 5. [SWI⁺] and [PIN⁺] prions demonstrate complementary interaction.

(A) Comparative analysis of the growth of strains containing combinations of [prion^-] or [PRION⁺] states for Rnq1 and Swi1 as well as deletions of the corresponding genes. “1”–The [SWI⁺][pin^-] and [swi^-][PIN⁺] strains were obtained from the 1-1-D931 [SWI⁺][PIN⁺] strain by deletion with subsequent reintroduction of RNQ1 and SWI1 genes, respectively (see “Materials and Methods”). The [swi^-][pin^-] strain was obtained from the 1-1-D931 [SWI⁺][PIN⁺] strain by GuHCl curing. “2”–The 26-1-4-1-1-D931 [swi^-][PIN⁺], 12-1-4-1-1-D931 [SWI⁺][pin^-], and 16-1-4-1-1-D931 [SWI⁺][PIN⁺] strains were obtained by transformation of the 1-4-1-1-D931 [swi^-][pin^-] recipient yeast cells with the 1-1-D931 [SWI⁺][PIN⁺] protein lysates followed by analysis of [SWI] and [PIN] status of the cells as described in “Materials and Methods”. Images were obtained after 5 days of incubation on–Ade plates with 150 μM CuSO₄ or after 3 passages on Gal plates. (B) Sedimentation analysis of Swi1(1–297)-YFP protein from the 16-1-4-1-1-D931 [SWI⁺][PIN⁺] and 12-1-4-1-1-D931 [SWI⁺][pin^-] strains expressing the pCUP1-SWI1(1–297)-YFP (URA3) plasmid. Soluble (S) and insoluble (I) fractions were obtained as indicated in Materials and Methods. Swi1(1–297)-YFP was detected using monoclonal rabbit primary antibodies against GFP [E385] (ab32146) (Abcam, Great Britain) and ECL Prime Western Blotting Detection Reagent kit (GE Healthcare, USA). (C) Analysis of the colocalization of Swi1-YFP and Rnq1-CFP aggregates. The cells of the D938 [SWI⁺][PIN⁺] strain were co-transformed with p426GPD–SWI1YFP and pCUP1-RNQ1-CFP(LEU2) plasmids. Transformants were selected on–Ura–Leu selective media with 150 μM CuSO₄ and incubated for 48 h prior to fluorescence microscopy.

Fig 6. [SWI⁺] and [PIN⁺] interaction causes nonsense suppression by decreasing SUP45 expression.

(A) Real-time PCR analysis of SUP45 mRNA levels in the [SWI⁺][PIN⁺], [SWI⁺][pin^-], and [swi^-][pin^-] strains. The results are presented as the 2^-ΔΔC(t) ± the standard deviation (for details, see Materials and Methods). Significance levels are indicated. (B) Images illustrating the differences in growth between [SWI⁺][PIN⁺], [SWI⁺][pin^-], and [swi^-][pin^-] strains on–Ade medium with 150 μM CuSO₄. Images were obtained after 5 days of incubation.

Fig 7. A scheme illustrating the influence of [SWI⁺] and [PIN⁺] interaction on nonsense suppression.

(A) Swi1 normally acts as an activator of SUP45 expression (black arrow). (B) Prion inactivation of Swi1 partially blocks activation of SUP45 expression resulting in weak nonsense suppression (dark grey arrow). (C) [PIN⁺] indirectly enhances Swi1 inactivation in the [SWI⁺] strain (light grey dashed line), thereby blocking the Swi1-dependent activation of SUP45 expression (light grey arrow).