Abstract
Human α-synuclein expression in baker’s yeast reportedly induces mitochondria-dependent apoptosis. Surprisingly, we find that, under de-repressing conditions of the inducible MET25/GAL1 promoters, yeast cells expressing chromosomally-integrated copies of the human α-synuclein gene are not killed, but spontaneously form respiration-deficient rho-minus (ρ−) petites. Although yeast cells can undergo cell death (apoptosis) from loss of mitochondrial function, they can also survive without functional mitochondria. Such cells are referred to as ρ0 or ρ− petites. This study reports that minimal expression of human α-synuclein in yeast, from MET25/GAL1 promoter, gives rise to ρ− petites. Interestingly, the full expression of α-synuclein, from the same promoters, in α-synuclein-triggered ρ− petites and also in ρ0 petites (produced by treating ρ+ cells with the mutagen ethidium bromide) initiates apoptosis. The percentages of petites increase with increasing α-synuclein gene copy-number. ρ− petites expressing α-synuclein from fully-induced MET25/GAL1 promoters exhibit increased ROS levels, loss of mitochondrial membrane potential, and nuclear DNA fragmentation, with increasing copies of α-synuclein. Our results indicate that, for the first time in yeast, α-synuclein-triggered apoptosis can occur independently of functional mitochondria. The observation that α-synuclein naturally forms petites and that they can undergo apoptosis may have important implications in understanding the pathogenesis of Parkinson’s disease.
Highlights
The yeast Saccharomyces cerevisiae is an attractive tool for the elucidation of human cells’ diverse biochemical pathways, which includes mitochondria-dependent apoptosis, a form of programmed cell death [1,2,3]
During the process of sequential chromosomal integration of MET25p or GAL1 Promoter (GAL1p)-driven human α-syn gene-expression cassette-bearing plasmids (Supplementary Materials, Parts 1 and 2) to obtain yeast strains containing 1, 2, and 3 copies of the α-syn gene, it was found, after 72 h incubation at 30 ◦ C, that a percentage of transformants did not grow on solid-agar complete YP medium plates that contained Glycerol as the sole carbon source (Figure 1C,D; non-shaded bars; the only exception, where all transformants grew on Glycerol, were cells containing 1-copy MET25p-α-syn)
The results obtained after full expression of α-syn in rho- and rho0 yeast cells showed some death in the petite cells; this indicates that mitochondrial function is not an absolute requirement for α-syn-mediated apoptosis
Summary
The yeast Saccharomyces cerevisiae is an attractive tool for the elucidation of human cells’ diverse biochemical pathways, which includes mitochondria-dependent apoptosis, a form of programmed cell death [1,2,3]. It has been reported that apoptosis was induced in aged yeast cells by human α-synuclein (α-syn) overproduction; it was thought to cause Parkinson’s disease (PD) in human neuronal cells (PD) [4], occurs in the presence of functional mitochondria [5]. In both yeast and human neurons, α-syn’s toxicity seems to be dependent on mitochondrial outer membrane regulator (VDAC) that controls the influx and efflux of metabolites in and out of the mitochondria [6].
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