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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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Htt103Q and α-syn toxicity is not suppressed in mRNA export mutants, whereas TDP-43 toxicity is suppressed. Tenfold serial dilutions of cells were spotted onto medium containing glucose (expression repressed) or galactose (expression induced) to measure spotting efficiency and toxicity of Htt25Q and Htt103Q (A), TDP-43 or α-syn (B) in wild-type and deletion mutant cells.
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Figure 10: Htt103Q and α-syn toxicity is not suppressed in mRNA export mutants, whereas TDP-43 toxicity is suppressed. Tenfold serial dilutions of cells were spotted onto medium containing glucose (expression repressed) or galactose (expression induced) to measure spotting efficiency and toxicity of Htt25Q and Htt103Q (A), TDP-43 or α-syn (B) in wild-type and deletion mutant cells.

Mentions: It could be that the mRNA nuclear export mutants are simply diminishing the amount of Pab117A mRNA in the cytoplasm and thus reducing Pab117A protein levels. The initial deletion screens were performed with the Pab117A gene integrated as one copy in the yeast genome. In our subsequent tests, we observed similar suppression of Pab117A when it was expressed from a multicopy plasmid (Figure 9A). Furthermore, none of the deletions suppressed Pab120A toxicity (Figure 9A), Htt103Q toxicity (Figure 10A), or α-syn toxicity (Figure 10B). Suppression of Pab120A, Htt103Q, or α-syn toxicity would have been expected if the effects of the deletions were simply to reduce mRNA levels to a point that disallowed sufficient expression.


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)

Htt103Q and α-syn toxicity is not suppressed in mRNA export mutants, whereas TDP-43 toxicity is suppressed. Tenfold serial dilutions of cells were spotted onto medium containing glucose (expression repressed) or galactose (expression induced) to measure spotting efficiency and toxicity of Htt25Q and Htt103Q (A), TDP-43 or α-syn (B) in wild-type and deletion mutant cells.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3113764&req=5

Figure 10: Htt103Q and α-syn toxicity is not suppressed in mRNA export mutants, whereas TDP-43 toxicity is suppressed. Tenfold serial dilutions of cells were spotted onto medium containing glucose (expression repressed) or galactose (expression induced) to measure spotting efficiency and toxicity of Htt25Q and Htt103Q (A), TDP-43 or α-syn (B) in wild-type and deletion mutant cells.
Mentions: It could be that the mRNA nuclear export mutants are simply diminishing the amount of Pab117A mRNA in the cytoplasm and thus reducing Pab117A protein levels. The initial deletion screens were performed with the Pab117A gene integrated as one copy in the yeast genome. In our subsequent tests, we observed similar suppression of Pab117A when it was expressed from a multicopy plasmid (Figure 9A). Furthermore, none of the deletions suppressed Pab120A toxicity (Figure 9A), Htt103Q toxicity (Figure 10A), or α-syn toxicity (Figure 10B). Suppression of Pab120A, Htt103Q, or α-syn toxicity would have been expected if the effects of the deletions were simply to reduce mRNA levels to a point that disallowed sufficient expression.

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