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. 1. 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 [1–4]. Each disease is characterized by neuronal death and accumulation of α-synuclein and several other proteins in cytoplasmic inclusions called Lewy bodies [5]. Of these, PD is the most prevalent disorder, afflicting over 4 million people worldwide [6]. While whether the aggregation of α-synuclein is neurotoxic or a protective cellular response is still being resolved, α-synuclein is intimately linked to pathogenesis as Lewy bodies are found in both sporadic and familial PD, and three point mutations (A53T, A30P, and E46K) within the α-synuclein gene itself cause PD [7–9]. Duplication or triplication of the α-synuclein locus also results in PD onset, further implicating α-synuclein in PD pathogenesis [10, 11]. Even sporadic PD is linked to polymorphisms in the α-synuclein gene that potentially increase protein production [12]. α-Synuclein is a short, highly flexible protein found throughout the brain and localized to presynaptic terminals of dopaminergic neurons [13–15]. Two biochemical properties of α-synuclein most clearly linked to PD pathology are intracellular
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