Contribution of Alanine-76 and Serine Phosphorylation inα-Synuclein Membrane Association and Aggregation in Yeasts

Michael Fiske,Shubhik Debburman,Rebecca Brezinsky,Keith Solvang,Michael White,Sara Herrera,Stephanie Valtierra,Alina Konnikova,Michael Zorniak

doi:10.4061/2011/392180

Abstract

In Parkinson's disease (PD), misfolded and aggregated α-synuclein protein accumulates in degenerating midbrain dopaminergic neurons. The amino acid alanine-76 in α-synuclein and phosphorylation at serine-87 and serine-129 are thought to regulate its aggregation and toxicity. However, their exact contributions to α-synuclein membrane association are less clear. We found that α-synuclein is indeed phosphorylated in fission yeast and budding yeast, the two models that we employed for assessing α-synuclein aggregation and membrane association properties, respectively. Surprisingly, blocking serine phosphorylation (S87A, S129A, and S87A/S129A) or mimicking it (S87D, S129D) altered α-synuclein aggregation in fission yeast. Either blocking or mimicking this phosphorylation increased endomembrane association in fission yeast, but only mimicking it decreased plasma membrane association in budding yeast. Polar substitution mutations of alanine-76 (A76E and A76R) decreased α-synuclein membrane association in budding yeast and decreased aggregation in fission yeast. These yeast studies extend our understanding of serine phosphorylation and alanine-76 contributions to α-synuclein aggregation and are the first to detail their impact on α-synuclein's plasma membrane and endomembrane association.

Highlights

The α-synucleinopathies are a group of neurodegenerative diseases that include Parkinson’s disease, dementia with Lewy bodies (DLB), multiple system atrophy (MSA), and Lewy body dysphagia [1,2,3,4]
Only one study has reported that wildtype (WT) α-synuclein is phosphorylated in a budding yeast model for studying Parkinson’s disease (PD)-related properties [46], but none have been reported for fission yeast
We referred to the αsynuclein localization pattern in fission yeasts as “aggregates” in our initial study [45] and do so again here, as they are distinct intracellular accumulations that are different from the plasma membrane or the vesicular membrane localization of α-synuclein-GFP in budding yeast

Summary

Introduction

The α-synucleinopathies are a group of neurodegenerative diseases that include Parkinson’s disease, dementia with Lewy bodies (DLB), multiple system atrophy (MSA), and Lewy body dysphagia [1,2,3,4]. Deletion of amino acids 71–82 within the NAC domain significantly decreases α-synuclein aggregation in vitro [19, 37] and in cell culture [38]. Our lab pioneered a fission yeast α-synuclein model in 2006, marking the first time Schizosaccharomyces pombe had been used to model PDassociated α-synuclein properties [45] These two yeast systems provide a unique research opportunity to comparatively study how α-synuclein exhibits aggregation and membrane association properties in each organism. Each yeast model recapitulates an important PD-related property of α-synuclein (aggregation or membrane association), allowing us to determine how serine phosphorylation and alanine-76 impact these two pathologically linked properties of α-synuclein. We found that alanine-76 contributed significantly to α-synuclein membrane association in budding yeast and regulated α-synuclein aggregation in fission yeast. Neither serine phosphorylation nor alanine significantly affected toxicity in either yeast model

Results

Discussion

Materials and Methods