Yeast cells provide insight into alpha-synuclein biology and pathobiology

Abstract

Alpha-synuclein is implicated in several neurodegenerative disorders, such as Parkinson's disease and multiple system atrophy, yet its functions remain obscure. When expressed in yeast, alpha-synuclein associated with the plasma membrane in a highly selective manner, before forming cytoplasmic inclusions through a concentration-dependent, nucleated process. Alpha-synuclein inhibited phospholipase D, induced lipid droplet accumulation, and affected vesicle trafficking. This readily manipulable system provides an opportunity to dissect the molecular pathways underlying normal alpha-synuclein biology and the pathogenic consequences of its misfolding.

Fig. 1

Expression of αSyn in yeast. (A) Immunoblot analysis of cells expressing αSyn-GFP fusions. Single integrated copies (left) of WT, A53T, and A30P αSyn produced similar amounts of protein (supporting online material). 2μ plasmid (variable copy number) expression levels of WT and A53T were similar but lower than those of A30P αSyn (right). (B) Fluorescence microscopy of yeast cells expressing αSyn-GFP. In cells carrying one copy of WT or A53T, the protein concentrated at the plasma membrane, and small amounts concentrated in the cytoplasm. Cells with two copies of WT or A53T showed cytoplasmic inclusions and reduced membrane localization. When expressed from the 2μ plasmid, WT and A53T distributions varied from cell to cell, whereas A30P showed a diffuse cytoplasmic distribution. (C) αSyn inhibits growth. One copy of αSyn WT or A53T had little effect on growth (left), whereas two copies completely inhibited it (middle). When expressed from a 2μ plasmid, A30P inhibited growth but less so than WT or A53T (right). (D) Immunofluorescence of cells expressing αSyn-GFP (2μ) (top) with an antibody to ubiquitin (bottom) showed increased levels of ubiquitin accumulation in cells expressing αSyn ( WT and both mutants). (E) Cells coexpressing αSyn and GFPu showed accumulation of the reporter when compared to cells carrying an empty vector control. Scale bars, 1 μm.

Fig. 2

αSyn inhibits PLD. A cki1Δ strain (top) or a sec14ts cki1Δ double-mutant strain (bottom) were transformed with 2μ plasmids expressing αSyn and grown at 37°C (supporting online material). WT and A53TαSyn blocked growth in sec14ts cki1Δ strain, but not in the cki1Δ strain.

Fig. 3

Cells expressing αSyn accumulate lipids. (A) Cells expressing either WT or A53T αSyn-GFP (top) were highly reactive for the lipophylic dye Nile red (bottom); cells expressing A30P were not (supporting online material). (B) Electron microscopy demonstrated the accumulation of lipid droplets (arrowheads) in cells expressing WT or A53T αSyn but not in cells expressing A30P. Scale bars, 1 μm.

Fig. 4

αSyn overexpression perturbs the distribution of vesicular pools. Endocytosis of the fluorescent dye FM4-64 (red) was used to monitor the effects of all three αSyn-GFP variants and of Q103 Htt exon 1 (green) on vesicular trafficking. Cells grown in raffinose (uninduced) show normal ring-like vacuolar staining (top). Expression of αSyn WT, A53T, and A30P markedly altered FM4-64 distributions (punctate structures in red, bottom). Scale bar, 1 μm.