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Posttranslational Modifications and Clearing of α-Synuclein Aggregates in Yeast.

Popova B, Kleinknecht A, Braus GH - Biomolecules (2015)

Bottom Line: Misfolded α-synuclein is one of their main constituents.The molecular mechanism involved in clearance of α-synuclein aggregates is a central question for elucidating the α-synuclein-related toxicity.Various modifications change α-synuclein posttranslationally and alter its inclusion formation, cytotoxicity and the distribution to different clearance pathways.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Microbiology and Genetics, Institute of Microbiology and Genetics, Georg-August-Universität Göttingen, D-37077 Göttingen, Germany. bpopova@gwdg.de.

ABSTRACT
The budding yeast Saccharomyces cerevisiae represents an established model system to study the molecular mechanisms associated to neurodegenerative disorders. A key-feature of Parkinson's disease is the formation of Lewy bodies, which are cytoplasmic protein inclusions. Misfolded α-synuclein is one of their main constituents. Expression of α-synuclein protein in yeast leads to protein aggregation and cellular toxicity, which is reminiscent to Lewy body containing human cells. The molecular mechanism involved in clearance of α-synuclein aggregates is a central question for elucidating the α-synuclein-related toxicity. Cellular clearance mechanisms include ubiquitin mediated 26S proteasome function as well as lysosome/vacuole associated degradative pathways as autophagy. Various modifications change α-synuclein posttranslationally and alter its inclusion formation, cytotoxicity and the distribution to different clearance pathways. Several of these modification sites are conserved from yeast to human. In this review, we summarize recent findings on the effect of phosphorylation and sumoylation of α-synuclein to the enhanced channeling to either the autophagy or the proteasome degradation pathway in yeast model of Parkinson's disease.

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Localization and impact of α-synuclein on yeast cell growth. (A) α-synuclein with C-terminally fused GFP is expressed from a regulatable galactose-inducible GAL1 promoter. GFP does not alter the aggregation behavior or the toxicity of the protein. (B) Time-dependent aggregate formation of α-synuclein, expressed from a high-copy plasmid, monitored by fluorescence microscopy. Indicated is the time after induction of expression: (a) early stage (1 h): α-synuclein is localized at the plasma membrane; (b) 2 h—Nucleation at the plasma membrane and formation of small membrane-connected aggregates; (c) late stage (6 h): Formation of large cytoplasmic aggregates by prolonged expression. (C) Growth behaviors of yeast cells, carrying increasing number of copies of GAL1-driven α-synuclein-GFP fusion alleles with different α-synuclein variants. Spotting analysis indicates decreased growth with increasing copy number of wild-type (WT) and A53T α-synuclein but not A30P. Scale bar = 1 µm; oe, overexpression.
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biomolecules-05-00617-f001: Localization and impact of α-synuclein on yeast cell growth. (A) α-synuclein with C-terminally fused GFP is expressed from a regulatable galactose-inducible GAL1 promoter. GFP does not alter the aggregation behavior or the toxicity of the protein. (B) Time-dependent aggregate formation of α-synuclein, expressed from a high-copy plasmid, monitored by fluorescence microscopy. Indicated is the time after induction of expression: (a) early stage (1 h): α-synuclein is localized at the plasma membrane; (b) 2 h—Nucleation at the plasma membrane and formation of small membrane-connected aggregates; (c) late stage (6 h): Formation of large cytoplasmic aggregates by prolonged expression. (C) Growth behaviors of yeast cells, carrying increasing number of copies of GAL1-driven α-synuclein-GFP fusion alleles with different α-synuclein variants. Spotting analysis indicates decreased growth with increasing copy number of wild-type (WT) and A53T α-synuclein but not A30P. Scale bar = 1 µm; oe, overexpression.

Mentions: A homologue of the SNCA gene encoding human α-synuclein is not present in the yeast genome. However, several relevant aspects of the Parkinson’s disease phenotype are recapitulated when different forms of human α-synuclein or even C-terminally tagged versions are heterologously expressed in yeast cells (Figure 1A; [54,59,62,63,64]). α-synuclein, as well as the mutant variant A53T, which had been discovered in inherited PD patients, are delivered to the yeast plasma membrane through the secretory pathway [56]. This is consistent with the known affinity of α-synuclein for phospholipids. Once the proteins accumulate there, they start to form small seeds that continue to grow in size (Figure 1B). Increase in the expression level of α-synuclein dramatically changes its localization and leads to formation of cytoplasmic inclusions, similar to LBs in neurons of Parkinson patients. This is accompanied with an increase in toxicity, defined as a reduction in cell growth followed by cell death. The increase in α-synuclein mediated toxicity is dose-dependent with a threshold for toxicity (Figure 1C). Three copies of wild-type α-synuclein and two copies of A53T α-synuclein, integrated into a single genomic locus were shown to cause growth inhibition and aggregate formation when the α-synuclein encoding gene was driven by a regulatable house-keeping promoter as the GAL1 promoter [63]. This promoter is normally required for the catabolism of galactose and is only activated when this sugar is available as nutrient. The response to α-synuclein expression is under these conditions similar to humans, where duplication or triplication of the SCNA gene locus driven by its own promoter causes early on-set of PD [9,65].


Posttranslational Modifications and Clearing of α-Synuclein Aggregates in Yeast.

Popova B, Kleinknecht A, Braus GH - Biomolecules (2015)

Localization and impact of α-synuclein on yeast cell growth. (A) α-synuclein with C-terminally fused GFP is expressed from a regulatable galactose-inducible GAL1 promoter. GFP does not alter the aggregation behavior or the toxicity of the protein. (B) Time-dependent aggregate formation of α-synuclein, expressed from a high-copy plasmid, monitored by fluorescence microscopy. Indicated is the time after induction of expression: (a) early stage (1 h): α-synuclein is localized at the plasma membrane; (b) 2 h—Nucleation at the plasma membrane and formation of small membrane-connected aggregates; (c) late stage (6 h): Formation of large cytoplasmic aggregates by prolonged expression. (C) Growth behaviors of yeast cells, carrying increasing number of copies of GAL1-driven α-synuclein-GFP fusion alleles with different α-synuclein variants. Spotting analysis indicates decreased growth with increasing copy number of wild-type (WT) and A53T α-synuclein but not A30P. Scale bar = 1 µm; oe, overexpression.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4496687&req=5

biomolecules-05-00617-f001: Localization and impact of α-synuclein on yeast cell growth. (A) α-synuclein with C-terminally fused GFP is expressed from a regulatable galactose-inducible GAL1 promoter. GFP does not alter the aggregation behavior or the toxicity of the protein. (B) Time-dependent aggregate formation of α-synuclein, expressed from a high-copy plasmid, monitored by fluorescence microscopy. Indicated is the time after induction of expression: (a) early stage (1 h): α-synuclein is localized at the plasma membrane; (b) 2 h—Nucleation at the plasma membrane and formation of small membrane-connected aggregates; (c) late stage (6 h): Formation of large cytoplasmic aggregates by prolonged expression. (C) Growth behaviors of yeast cells, carrying increasing number of copies of GAL1-driven α-synuclein-GFP fusion alleles with different α-synuclein variants. Spotting analysis indicates decreased growth with increasing copy number of wild-type (WT) and A53T α-synuclein but not A30P. Scale bar = 1 µm; oe, overexpression.
Mentions: A homologue of the SNCA gene encoding human α-synuclein is not present in the yeast genome. However, several relevant aspects of the Parkinson’s disease phenotype are recapitulated when different forms of human α-synuclein or even C-terminally tagged versions are heterologously expressed in yeast cells (Figure 1A; [54,59,62,63,64]). α-synuclein, as well as the mutant variant A53T, which had been discovered in inherited PD patients, are delivered to the yeast plasma membrane through the secretory pathway [56]. This is consistent with the known affinity of α-synuclein for phospholipids. Once the proteins accumulate there, they start to form small seeds that continue to grow in size (Figure 1B). Increase in the expression level of α-synuclein dramatically changes its localization and leads to formation of cytoplasmic inclusions, similar to LBs in neurons of Parkinson patients. This is accompanied with an increase in toxicity, defined as a reduction in cell growth followed by cell death. The increase in α-synuclein mediated toxicity is dose-dependent with a threshold for toxicity (Figure 1C). Three copies of wild-type α-synuclein and two copies of A53T α-synuclein, integrated into a single genomic locus were shown to cause growth inhibition and aggregate formation when the α-synuclein encoding gene was driven by a regulatable house-keeping promoter as the GAL1 promoter [63]. This promoter is normally required for the catabolism of galactose and is only activated when this sugar is available as nutrient. The response to α-synuclein expression is under these conditions similar to humans, where duplication or triplication of the SCNA gene locus driven by its own promoter causes early on-set of PD [9,65].

Bottom Line: Misfolded α-synuclein is one of their main constituents.The molecular mechanism involved in clearance of α-synuclein aggregates is a central question for elucidating the α-synuclein-related toxicity.Various modifications change α-synuclein posttranslationally and alter its inclusion formation, cytotoxicity and the distribution to different clearance pathways.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Microbiology and Genetics, Institute of Microbiology and Genetics, Georg-August-Universität Göttingen, D-37077 Göttingen, Germany. bpopova@gwdg.de.

ABSTRACT
The budding yeast Saccharomyces cerevisiae represents an established model system to study the molecular mechanisms associated to neurodegenerative disorders. A key-feature of Parkinson's disease is the formation of Lewy bodies, which are cytoplasmic protein inclusions. Misfolded α-synuclein is one of their main constituents. Expression of α-synuclein protein in yeast leads to protein aggregation and cellular toxicity, which is reminiscent to Lewy body containing human cells. The molecular mechanism involved in clearance of α-synuclein aggregates is a central question for elucidating the α-synuclein-related toxicity. Cellular clearance mechanisms include ubiquitin mediated 26S proteasome function as well as lysosome/vacuole associated degradative pathways as autophagy. Various modifications change α-synuclein posttranslationally and alter its inclusion formation, cytotoxicity and the distribution to different clearance pathways. Several of these modification sites are conserved from yeast to human. In this review, we summarize recent findings on the effect of phosphorylation and sumoylation of α-synuclein to the enhanced channeling to either the autophagy or the proteasome degradation pathway in yeast model of Parkinson's disease.

Show MeSH
Related in: MedlinePlus