α-Synuclein Targets the Plasma Membrane via the Secretory Pathway and Induces Toxicity in Yeast

Abstract

A pathological feature of Parkinson's disease is the presence of Lewy bodies within selectively vulnerable neurons. These are ubiquitinated cytoplasmic inclusions containing ␣-synuclein, an abundant protein normally associated with presynaptic terminals. Point mutations in the ␣-synuclein gene (A30P and A53T), as well as triplication of the wild-type (WT) locus, have been linked to autosomal dominant Parkinson's. How these alterations might contribute to disease progression is unclear. Using the genetically tractable yeast Saccharomyces cerevisiae as a model system, we find that both the WT and the A53T isoforms of ␣-synuclein initially localize to the plasma membrane, to which they are delivered via the classical secretory pathway. In contrast, the A30P mutant protein disperses within the cytoplasm and does not associate with the plasma membrane, and its intracellular distribution is unaffected by mutations in the secretory pathway. When their expression is elevated, WT and A53T, but not A30P, are toxic to cells. At moderate levels of expression, WT and A53T induce the cellular stress (heat-shock) response and are toxic to cells bearing mutations in the 20S proteasome. Our results reveal a link between plasma membrane targeting of ␣-synuclein and its toxicity in yeast and suggest a role for the quality control (QC) system in the cell's effort to deal with this natively unfolded protein.

Relationships between the Sequence of α-Synuclein and its Membrane Affinity, Fibrillization Propensity, and Yeast Toxicity

“…In conclusion, we support a model of yeast toxicity in which overexpressed monomeric synuclein directly coats the plasma and internal membranes via its amphipathic α-helices, disrupting normal membrane processes (including vesicle trafficking [19][20][21], and eventually leading to toxicity. Perhaps the simplest mechanism by which synuclein binding could disrupt membrane processes is a non-specific one.…”

“…18,19 Wild type and A53T α-synuclein are both toxic and localize to inclusions and the plasma membrane when expressed from multiple GAL promoters, such as those on 2-micron plasmids (high copy number) or from multiple integrated copies. 18,20,21 In contrast, the A30P variant is non-toxic to yeast and is uniformly distributed throughout the cytoplasm. 18 All of the synuclein proteins are relatively non-toxic when expressed from a single copy.…”

“…1; discussed below). Dixon et al 21 showed that mutation of proteins in the yeast secretory pathway (including ER-Golgi transport) can cause α-synuclein to change its localization from plasma membrane to punctate inclusions. It was later shown that α-synuclein itself can disrupt ER-Golgi trafficking.…”

Domain a’ of protein disulfide isomerase plays key role in inhibiting α-synuclein fibril formation

“…In immunogold electron microscopy examination of rat brain neurons, the αSyn concentration was found to be higher in endoplasmic reticulum (ER) than in cytoplasmic region (Zhang et al 2008). Synthesized αSyn in yeast was found to be translocated from the ER to the Golgi and further to the plasma membrane through a classical secretion pathway (Dixon et al 2005). Recently, αSyn has been reported to be involved in the impairment of ER functions.…”

Splice isoform and pharmacological studies reveal that sterol depletion relocalizes α-synuclein and enhances its toxicity

“…Much of what now is known about these processes was discovered first in yeast and later applied to cells of higher organisms, including neurons. In yeast, however, secretion is constitutive, allowing vesicles to fuse with the plasma membrane without an external signal (15). In neurons, in contrast, presynaptic vesicles fuse with the plasma membrane in a controlled manner.…”