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A yeast model for polyalanine-expansion aggregation and toxicity.

Konopka CA, Locke MN, Gallagher PS, Pham N, Hart MP, Walker CJ, Gitler AD, Gardner RG - Mol. Biol. Cell (2011)

Bottom Line: Nine human disorders result from the toxic accumulation and aggregation of proteins with expansions in their endogenous polyalanine (polyA) tracts.In our initial case, we expanded the polyA tract within the native yeast poly(Adenine)-binding protein Pab1 from 8A to 13A, 15A, 17A, and 20A.Surprisingly, neither manipulation suppressed the cytotoxicity of 20A-expanded Pab1.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, University of Washington, Seattle, USA.

ABSTRACT
Nine human disorders result from the toxic accumulation and aggregation of proteins with expansions in their endogenous polyalanine (polyA) tracts. Given the prevalence of polyA tracts in eukaryotic proteomes, we wanted to understand the generality of polyA-expansion cytotoxicity by using yeast as a model organism. In our initial case, we expanded the polyA tract within the native yeast poly(Adenine)-binding protein Pab1 from 8A to 13A, 15A, 17A, and 20A. These expansions resulted in increasing formation of Pab1 inclusions, insolubility, and cytotoxicity that correlated with the length of the polyA expansion. Pab1 binds mRNA as part of its normal function, and disrupting RNA binding or altering cytoplasmic mRNA levels suppressed the cytotoxicity of 17A-expanded Pab1, indicating a requisite role for mRNA in Pab1 polyA-expansion toxicity. Surprisingly, neither manipulation suppressed the cytotoxicity of 20A-expanded Pab1. Thus longer expansions may have a different mechanism for toxicity. We think that this difference underscores the potential need to examine the cytotoxic mechanisms of both long and short expansions in models of expansion disorders.

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Pab117A but not Pab120A toxicity is suppressed in mRNA export mutants. (A) Tenfold serial dilutions of cells were spotted onto medium containing glucose (expression repressed) r galactose (expression induced) to measure spotting efficiency toxicity of Pab18A-GFP, Pab117A-GFP, or Pab120A-GFP in wild-type and deletion mutant cells. (B) Wild-type and deletion mutant cells expressing Pab18A-GFP, Pab117A-GFP, or Pab120A-GFP were assayed for presence of GFP-positive inclusions and morphology defects after 16-h induction and reported as a percentage of GFP-positive cells. More than 150 cells in three independent cultures were counted for each. Bars are SD. *p < 0.05 by Student's t test when polyA-expansion Pab1 mutant strains were compared with corresponding wild type.
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Figure 9: Pab117A but not Pab120A toxicity is suppressed in mRNA export mutants. (A) Tenfold serial dilutions of cells were spotted onto medium containing glucose (expression repressed) r galactose (expression induced) to measure spotting efficiency toxicity of Pab18A-GFP, Pab117A-GFP, or Pab120A-GFP in wild-type and deletion mutant cells. (B) Wild-type and deletion mutant cells expressing Pab18A-GFP, Pab117A-GFP, or Pab120A-GFP were assayed for presence of GFP-positive inclusions and morphology defects after 16-h induction and reported as a percentage of GFP-positive cells. More than 150 cells in three independent cultures were counted for each. Bars are SD. *p < 0.05 by Student's t test when polyA-expansion Pab1 mutant strains were compared with corresponding wild type.

Mentions: Last, we wanted to take advantage of yeast's powerful genetic methods to identify modifiers of Pab1 polyA-expansion toxicity. We were particularly interested in genetic mutations that could suppress toxicity. Therefore we used a strain in which Pab117A was expressed from a single genomically inserted copy of the PAB117A to screen the yeast MATa deletion collection for individual gene deletions that suppressed the growth defect caused by Pab117A expression. We independently conducted the screen three times and selected deletion strains that reproduced suppression of the Pab117A growth defect each time. From the independent screens, we identified 10 gene deletions that consistently suppressed the Pab117A growth defect (thp2Δ, mft1Δ, ynl140cΔ, sac3Δ, ade4Δ, ade8Δ, sse1Δ, sur4Δ, ptk2Δ, arg80Δ). Each putative suppressor that passed these criteria was recapitulated in an independent yeast strain and retested for suppression of Pab117A toxicity. Of these, only thp2Δ, mft1Δ, and sac3Δ suppressed the Pab117A growth defect (Figure 9A). Interestingly, none of these deletions suppressed the Pab120A growth defect (Figure 9A), once again indicating a profound difference between the toxicity of Pab117A compared with Pab120A.


A yeast model for polyalanine-expansion aggregation and toxicity.

Konopka CA, Locke MN, Gallagher PS, Pham N, Hart MP, Walker CJ, Gitler AD, Gardner RG - Mol. Biol. Cell (2011)

Pab117A but not Pab120A toxicity is suppressed in mRNA export mutants. (A) Tenfold serial dilutions of cells were spotted onto medium containing glucose (expression repressed) r galactose (expression induced) to measure spotting efficiency toxicity of Pab18A-GFP, Pab117A-GFP, or Pab120A-GFP in wild-type and deletion mutant cells. (B) Wild-type and deletion mutant cells expressing Pab18A-GFP, Pab117A-GFP, or Pab120A-GFP were assayed for presence of GFP-positive inclusions and morphology defects after 16-h induction and reported as a percentage of GFP-positive cells. More than 150 cells in three independent cultures were counted for each. Bars are SD. *p < 0.05 by Student's t test when polyA-expansion Pab1 mutant strains were compared with corresponding wild type.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3113764&req=5

Figure 9: Pab117A but not Pab120A toxicity is suppressed in mRNA export mutants. (A) Tenfold serial dilutions of cells were spotted onto medium containing glucose (expression repressed) r galactose (expression induced) to measure spotting efficiency toxicity of Pab18A-GFP, Pab117A-GFP, or Pab120A-GFP in wild-type and deletion mutant cells. (B) Wild-type and deletion mutant cells expressing Pab18A-GFP, Pab117A-GFP, or Pab120A-GFP were assayed for presence of GFP-positive inclusions and morphology defects after 16-h induction and reported as a percentage of GFP-positive cells. More than 150 cells in three independent cultures were counted for each. Bars are SD. *p < 0.05 by Student's t test when polyA-expansion Pab1 mutant strains were compared with corresponding wild type.
Mentions: Last, we wanted to take advantage of yeast's powerful genetic methods to identify modifiers of Pab1 polyA-expansion toxicity. We were particularly interested in genetic mutations that could suppress toxicity. Therefore we used a strain in which Pab117A was expressed from a single genomically inserted copy of the PAB117A to screen the yeast MATa deletion collection for individual gene deletions that suppressed the growth defect caused by Pab117A expression. We independently conducted the screen three times and selected deletion strains that reproduced suppression of the Pab117A growth defect each time. From the independent screens, we identified 10 gene deletions that consistently suppressed the Pab117A growth defect (thp2Δ, mft1Δ, ynl140cΔ, sac3Δ, ade4Δ, ade8Δ, sse1Δ, sur4Δ, ptk2Δ, arg80Δ). Each putative suppressor that passed these criteria was recapitulated in an independent yeast strain and retested for suppression of Pab117A toxicity. Of these, only thp2Δ, mft1Δ, and sac3Δ suppressed the Pab117A growth defect (Figure 9A). Interestingly, none of these deletions suppressed the Pab120A growth defect (Figure 9A), once again indicating a profound difference between the toxicity of Pab117A compared with Pab120A.

Bottom Line: Nine human disorders result from the toxic accumulation and aggregation of proteins with expansions in their endogenous polyalanine (polyA) tracts.In our initial case, we expanded the polyA tract within the native yeast poly(Adenine)-binding protein Pab1 from 8A to 13A, 15A, 17A, and 20A.Surprisingly, neither manipulation suppressed the cytotoxicity of 20A-expanded Pab1.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, University of Washington, Seattle, USA.

ABSTRACT
Nine human disorders result from the toxic accumulation and aggregation of proteins with expansions in their endogenous polyalanine (polyA) tracts. Given the prevalence of polyA tracts in eukaryotic proteomes, we wanted to understand the generality of polyA-expansion cytotoxicity by using yeast as a model organism. In our initial case, we expanded the polyA tract within the native yeast poly(Adenine)-binding protein Pab1 from 8A to 13A, 15A, 17A, and 20A. These expansions resulted in increasing formation of Pab1 inclusions, insolubility, and cytotoxicity that correlated with the length of the polyA expansion. Pab1 binds mRNA as part of its normal function, and disrupting RNA binding or altering cytoplasmic mRNA levels suppressed the cytotoxicity of 17A-expanded Pab1, indicating a requisite role for mRNA in Pab1 polyA-expansion toxicity. Surprisingly, neither manipulation suppressed the cytotoxicity of 20A-expanded Pab1. Thus longer expansions may have a different mechanism for toxicity. We think that this difference underscores the potential need to examine the cytotoxic mechanisms of both long and short expansions in models of expansion disorders.

Show MeSH
Related in: MedlinePlus