Apoptosis, Induced by Human α-Synuclein in Yeast, Can Occur Independent of Functional Mitochondria

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.


PART 1: Construction of Yeast Strains that Contain 1-copy, 2-and 3-Copies of the Human α-Syn Gene Driven by the MET25 Promoter (MET25p)
Plasmids were constructed that allow expression of 1-copy, 2-and 3-copies of the α-syn gene in yeast from the MET25 promoter (MET25p). A SpeI-SalI fragment of the coding sequence of the human α-syn gene [NCBI Accession # NM_000345.3] was isolated from a human hippocampal cDNA library (BioCat) by PCR and was cloned in yeast integration vectors downstream of the MET25p [Saccharomyces Genome Database ID S000004294] and upstream of the CYC1 gene terminator signal [Saccharomyces Genome Database ID S000003809]. The MET25 promoter is repressed in the presence of methionine and induced in its absence. After cloning of the α-syn gene in appropriate yeast integrative vectors, the following plasmids were obtained: YIpHIS3MET25p/alpha-syn (A), YIpURA3MET25p/alpha-syn (B), and YIpTRP1MET25p/alpha-syn (C) ( Figure S1). These plasmids contain the α-syn gene sandwiched between the MET25 promoter and CYC1 terminator signal and allow integration of 1-copy, 2-copies, and 3-copies of the α-syn gene into chromosomal locations where the auxotrophic markers, TRP1, HIS3, and URA3 genes, reside on the yeast genome.
The basic yeast strains used for integration were ρ + W303-1a (Mata, ade2, his3, leu2, trp1, ura3) (ATCC #208352) and its ρ 0 derivatives generated by treatment with ethidium bromide. Herein, the strains are referred to as BC300 or BC300-ρ + and BC300-ρ 0 . The 1-copy strains contain the TRP1 integrant. The 2-copy strains contain integrated copies of α-syn at the TRP1 and HIS3 loci, whereas the 3-copy strains contain integrated copies of α-syn at the TRP1, HIS3, and URA3 loci. To generate negative controls, the strains BC300-ρ + and BC300-ρ 0 were integrated with empty vectors (i.e., basic integrating vectors which do not contain the α-syn gene) in three successive steps to obtain the 6 strains:  The three integrative plasmids used to introduce human α-syn gene expression cassettes, under the control of the MET25 promoter, into three different chromosomal locations (i.e., where the TRP1, HIS3, URA3 genes lie) of the yeast genome. The arrows show the restriction sites at which the plasmids were linearized for genomic (i.e., chromosomal) integration via homologous recombination [1]. The restriction sites shown occur only once in the plasmid.

PART 2: Construction of Yeast Strains that Contain 1-copy, 2-and 3-Copies of the Human α-Syn Gene Driven by the GAL1 Promoter (GAL1p)
Plasmids were constructed that allow expression of 1-copy, 2-and 3-copies of the α-syn gene in yeast. A BglII-XbaI fragment of the coding sequence of the human α-syn gene [NCBI Accession # NM_000345.3] was isolated from a human hippocampal cDNA library (BioCat) by PCR and was cloned in yeast integration vectors downstream of the GAL1 promoter [Saccharomyces Genome Database ID S000000224] and upstream of the SUC2 gene terminator signal [Saccharomyces Genome Database ID S000001424]. The GAL1 promoter is repressed in the presence of glucose and induced in the presence of the sugar, galactose After cloning of the α-syn gene in appropriate yeast integrative vectors, the following plasmids were obtained: YIpHIS3GAL1p/alpha-syn YIpURA3GAL1p/alpha-syn (B), and YIpTRP1GAL1p/alpha-syn (C) ( Figure S2). These plasmids contain the α-syn gene sandwiched between the GAL1 promoter and SUC2 terminator signal and allow integration of one copy, two copies and three copies of the α-syn gene into chromosomal locations where the auxotrophic markers, TRP1, HIS3, and URA3 genes, reside on the yeast genome. The basic yeast strain used for integration was BC300. The 1-copy strain contains the TRP1 integrant. The 2-copy strain contains integrated copies of α-syn at the TRP1 and HIS3 loci, whereas the 3-copy strain contains integrated copies of α-syn at the TRP1, HIS3, and URA3 loci. To generate negative controls, the strain BC300 was integrated with empty vectors (i.e., basic integrating vectors which do not contain the α-syn gene) in three successive steps to obtain the three strains: (a) BC300::-(TRP1);(b) BC300::-(TRP1),-(URA3); and (c) BC300::-(TRP1),-(HIS3),-(URA3); they contain one copy, two and three copies of an empty plasmid at the (i) TRP1, (ii) TRP1 and HIS3, and (iii) TRP1, HIS3 and URA3, chromosomal loci. Figure 2. The three integrative plasmids used to introduce human α-syn gene expression cassettes, under the control of the GAL1 promoter, into three different chromosomal locations (i.e., where the TRP1, HIS3, URA3 genes lie) of the yeast genome. The arrows show the restriction sites at which the plasmids were linearized for genomic (i.e., chromosomal) integration via homologous recombination [1]. The restriction sites shown occur only once in the plasmid.

Copies of α-Syn Gene from MET25p, in ρ -Petite Yeast Cells
This densitometric quantification represents the Western blot depicted in Figure 2G of the manuscript. It clearly shows that the 3-copy strain expresses higher levels of α-syn than the 2-copy strain. Figure 2G) that show expression of α-syn protein from cells that express of 2, 3 copies of the α-syn gene from the MET25 promoter in ρpetite yeast cells. The values for β-actin (not shown) were roughly the same. The values represent the average of three independent experiments (p<0.05).

Copies of α-Syn Gene from GAL1p, in ρ -Petite Yeast Cells
This densitometric quantification represents the Western blot depicted in Figure 2H of the manuscript. It shows that there is a gradual increase of α-syn protein levels expressed in cells harbouring 1 to 3-copies of the α-syn gene.  Figure 2H) that show expression of α-syn protein from cells that express of 1, 2, 3 copies of the α-syn gene from the GAL1 promoter in ρpetite yeast cells. The values for β-actin (not shown) were roughly the same. The values represent the average of three independent experiments (p<0.05).