Limits...
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.

Show MeSH

Related in: MedlinePlus

Pab1 polyA-expansion inclusions are not P-bodies or stress granules. (A) Pab18A-GFP, Pab117A-GFP, or Pab120A-GFP expression was induced for 16 h in the indicated strains defective in P-body (edc3Δlsm4Δc) or stress granule formation (pub1Δ and pbp1Δ) and imaged with epifluorescence microscopy. (B) As in (A), except Pab18A-GFP, Pab117A-GFP, or Pab120A-GFP was coexpressed with Edc3-mCherry in wild-type cells. Bars = 2 μm.
© Copyright Policy - creative-commons
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3113764&req=5

Figure 4: Pab1 polyA-expansion inclusions are not P-bodies or stress granules. (A) Pab18A-GFP, Pab117A-GFP, or Pab120A-GFP expression was induced for 16 h in the indicated strains defective in P-body (edc3Δlsm4Δc) or stress granule formation (pub1Δ and pbp1Δ) and imaged with epifluorescence microscopy. (B) As in (A), except Pab18A-GFP, Pab117A-GFP, or Pab120A-GFP was coexpressed with Edc3-mCherry in wild-type cells. Bars = 2 μm.

Mentions: We wanted to determine whether the inclusions associated with the longer polyA tract expansions were new entities for Pab1 or whether they were previously defined structures in which Pab1 is known to be a component, such as P-bodies or stress granules (Buchan et al., 2008). P-bodies are dynamic inclusions that contain translationally silenced mRNAs and are thought to be involved in mRNA storage, translation repression, mRNA decapping, and nonsense-mediated decay (Parker and Sheth, 2007). Stress granules are also dynamic structures that contain untranslated mRNAs and form in response to translation initiation defects (Bond, 2006). To test whether Pab1 polyA-expansion inclusions are distinct from these defined cytoplasmic structures, Pab117A and Pab120A were expressed in yeast cells containing particular gene deletions (edc3Δlsm4ΔC, pub1Δ, or pbp1Δ) that render them defective in P-body or stress granule formation (Buchan et al., 2008). Pab117A-GFP and Pab120A-GFP formed inclusions in edc3Δ lsm4ΔC, pub1Δ, and pbp1Δ cells (Figure 4A), indicating that polyA-expansion–dependent inclusion formation still occurred in the absence of P-body or stress granule formation. Furthermore, the Pab117A- or Pab120A-positive inclusions did not colocalize with P-bodies marked by Edc3-GFP (Figure 4B). Thus Pab1 polyA-expansion inclusions are previously unknown cytoplasmic structures induced by the properties of the expanded polyA tract.


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)

Pab1 polyA-expansion inclusions are not P-bodies or stress granules. (A) Pab18A-GFP, Pab117A-GFP, or Pab120A-GFP expression was induced for 16 h in the indicated strains defective in P-body (edc3Δlsm4Δc) or stress granule formation (pub1Δ and pbp1Δ) and imaged with epifluorescence microscopy. (B) As in (A), except Pab18A-GFP, Pab117A-GFP, or Pab120A-GFP was coexpressed with Edc3-mCherry in wild-type cells. Bars = 2 μm.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 4: Pab1 polyA-expansion inclusions are not P-bodies or stress granules. (A) Pab18A-GFP, Pab117A-GFP, or Pab120A-GFP expression was induced for 16 h in the indicated strains defective in P-body (edc3Δlsm4Δc) or stress granule formation (pub1Δ and pbp1Δ) and imaged with epifluorescence microscopy. (B) As in (A), except Pab18A-GFP, Pab117A-GFP, or Pab120A-GFP was coexpressed with Edc3-mCherry in wild-type cells. Bars = 2 μm.
Mentions: We wanted to determine whether the inclusions associated with the longer polyA tract expansions were new entities for Pab1 or whether they were previously defined structures in which Pab1 is known to be a component, such as P-bodies or stress granules (Buchan et al., 2008). P-bodies are dynamic inclusions that contain translationally silenced mRNAs and are thought to be involved in mRNA storage, translation repression, mRNA decapping, and nonsense-mediated decay (Parker and Sheth, 2007). Stress granules are also dynamic structures that contain untranslated mRNAs and form in response to translation initiation defects (Bond, 2006). To test whether Pab1 polyA-expansion inclusions are distinct from these defined cytoplasmic structures, Pab117A and Pab120A were expressed in yeast cells containing particular gene deletions (edc3Δlsm4ΔC, pub1Δ, or pbp1Δ) that render them defective in P-body or stress granule formation (Buchan et al., 2008). Pab117A-GFP and Pab120A-GFP formed inclusions in edc3Δ lsm4ΔC, pub1Δ, and pbp1Δ cells (Figure 4A), indicating that polyA-expansion–dependent inclusion formation still occurred in the absence of P-body or stress granule formation. Furthermore, the Pab117A- or Pab120A-positive inclusions did not colocalize with P-bodies marked by Edc3-GFP (Figure 4B). Thus Pab1 polyA-expansion inclusions are previously unknown cytoplasmic structures induced by the properties of the expanded polyA tract.

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