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Apoptosis is a form of programmed cell death which is essential for the growth of dividing human cells whereas, in contrast, it is deleterious for post-mitotic cells such as neurons. Bax and α-synuclein are two human proteins which play a role in the induction of neuronal apoptosis in neurodegenerative diseases like Alzheimer's and Parkinson's. Human Bax and α-synuclein also induce cell death when expressed in baker's yeast, Saccharomyces cerevisiae. Quite unexpectedly, the human α-synuclein gene had been identified as an inhibitor of pro-apoptotic Bax using a yeast-based screen of a human hippocampal cDNA library. Plasmids were constructed with different promoters, which allow expression of wildtype and Parkinson's disease (PD)-related mutant α-synuclein genes, from (i) multi-copy 2µ (episomal) plasmids and (ii) integrative plasmids that compel expression of genes from chromosomal sites in varying copy numbers (1-3). All α-synuclein-containing plasmids were introduced, through transformation, into a yeast strain which already contained a chromosomally integrated copy of Bax. It is for the first time that it was observed that, depending on gene dosage, only wildtype α-synuclein is anti-apoptotic while mutant α-synuclein is not. The results also indicate that wildtype α-synuclein has a remarkable ability to manifest two contrasting effects depending on its level of expression: (i) normally, it would negate apoptosis but (ii) when overexpressed, it tends to induce apoptosis which is probably what happens in PD., (© 2020 The Author(s).)

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 ρ 0 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 ρ 0 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.

Eukaryotic elongation factor 1A1 (eEF1A1) is central to translational activity. It is involved in complexes that form signal transduction with protein kinase C, as well as being a signal transducer and activator of transcription 3. eEF1A1 and eEF1A2 are isoforms of the alpha subunit of elongating factor 1 complex. It has been reported that eEF1A1 is expressed in most human tissues but the brain, skeletal muscle and heart. eEF1A1 has been linked to both apoptosis and anti-apoptotic activities. In this study, eEF1A1 was co-expressed with Bax, a proapoptotic protein via heterologous expression of recombinant DNA in yeast cells. Assays were carried out to monitor the fate and state of yeast cells when eEF1A1 was co-expressed with Bax. The yeast strain (bearing an integrated copy of the Bax gene) was transformed with an episomal 2-micron plasmid that encodes HA-tagged eEF1A1 gene. The resultant strain would allow co-expression of Bax and eEF1A1 in yeast cells, Bax being under the control of the GAL1 promoter, while the PGK1 promoter drives eEF1A1 expression. Bcl 2A1, a known anti-apoptotic protein, was also co-expressed with Bax in yeast cells as a positive control, to study the anti-apoptotic characteristic of eEF-1A1. The part eEF1A1 plays in apoptosis has been contentious, amidst the pro and anti-apoptotic properties of eEF1A1, it was shown clearly, in this study that eEF1A1 portrays only anti-apoptotic property in the presence of pro-apoptotic protein, Bax.