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Proteome-wide search for PP2A substrates in fission yeast
Manuel Bernal1, Jacob Zhurinsky, Ana B Iglesias-Romero
1Centro Andaluz de Biología del Desarrollo, Universidad Pablo de Olavide-Consejo Superior de Investigaciones Científicas, Junta de Andalucía, Sevilla, Spain.
Abstract:
PP2A (protein phosphatase 2A) is a major phosphatase in eukaryotic cells that plays an essential role in many processes. PP2A mutations in Schizosaccharomyces pombe result in defects of cell cycle control, cytokinesis and morphogenesis. Which PP2A substrates are responsible for these changes is not known. In this work, we searched for PP2A substrates in S. pombe using two approaches, 2D-DIGE analysis of PP2A complex mutants and identification of PP2A interacting proteins. In both cases, we used MS to identify proteins of interest. In the DIGE experiment, we compared proteomes of wild-type S. pombe, deletion of pta2, the phosphoactivator of the PP2A catalytic subunit, and pab1-4, a mutant of B-type PP2A regulatory subunit. A total of 1742 protein spots were reproducibly resolved by 2D-DIGE and 51 spots demonstrated significant changes between PP2A mutants and the wild-type control. MS analysis of these spots identified 27 proteins that include key regulators of glycerol synthesis, carbon metabolism, amino acid biosyntesis, vitamin production, and protein folding. Importantly, we independently identified a subset of these proteins as PP2A binding partners by affinity precipitation, suggesting they may be direct targets of PP2A. We have validated our approach by demonstrating that phosphorylation of Gpd1, a key enzyme in glycerol biogenesis, is regulated by PP2A and that ability of cells to respond to osmotic stress by synthesizing glycerol is compromised in the PP2A mutants. Our work contributes to a better understanding of PP2A function and identifies potential PP2A substrates.
Insights
Protein phosphatase 2A (PP2A) mutations in yeast cause cell cycle defects. This study identifies key PP2A enzyme substrates involved in glycerol synthesis and osmotic stress response, advancing understanding of PP2A function.
Area of Science:
- Cell Biology
- Molecular Biology
- Biochemistry
Background:
- Protein phosphatase 2A (PP2A) is crucial for eukaryotic cellular processes.
- PP2A mutations in Schizosaccharomyces pombe lead to cell cycle, cytokinesis, and morphogenesis defects.
- The specific PP2A substrates responsible for these cellular defects remain largely unknown.
Purpose of the Study:
- To identify novel protein phosphatase 2A (PP2A) substrates in Schizosaccharomyces pombe.
- To elucidate the role of PP2A in regulating cellular processes through substrate identification.
- To understand the molecular mechanisms underlying PP2A-mediated cellular control.
Main Methods:
- Utilized 2D-DIGE analysis comparing wild-type and PP2A complex mutants (pta2 deletion, pab1-4 mutant).
- Employed mass spectrometry (MS) to identify proteins exhibiting altered expression in PP2A mutants.
- Validated PP2A-protein interactions using affinity precipitation and confirmed substrate regulation through phosphorylation analysis.
Main Results:
- Identified 27 potential PP2A substrates, including regulators of glycerol synthesis, carbon metabolism, amino acid biosynthesis, vitamin production, and protein folding.
- Confirmed a subset of these proteins as PP2A binding partners, suggesting direct substrate-enzyme interactions.
- Demonstrated PP2A regulation of Gpd1 phosphorylation, impacting glycerol synthesis and osmotic stress response in yeast.
Conclusions:
- This study successfully identified novel substrates of protein phosphatase 2A (PP2A) in S. pombe.
- The findings highlight PP2A's role in regulating metabolic pathways, particularly glycerol synthesis and osmotic stress response.
- Provides a foundation for further research into PP2A's diverse cellular functions and substrate interactions.
