Rescue of Bax-induced block of cell growth by α-synuclein-HA expressed from 1 to 3 copies of integrated plasmid, under the control of GAL1 promoter
(A) Growth of BC300::Bax yeast cells harbouring 1 copy of α-synuclein-HA gene (1) and 1 copy control (2), 2 copies of the α-synuclein-HA gene (3) and 2 copies control (4) and 3 copies of the α-synuclein-HA gene (5) and 3 copies control (6). Cells were streaked out on to two minimal medium plates, SD-Agar (containing glucose, plate A) and SG-Agar (containing galactose; plate B); plates were incubated at 30°C for 96 h. (B) Growth of BC300::Bax cells containing 1 copy, 2 or 3 copies of α-synuclein-HA and controls. Cells were incubated in galactose-containing minimal liquid medium for 48 h. The results show that 2 copies of HA-tagged α-synuclein can rescue Bax-induced block in cell growth. ANOVA test was performed which revealed that the difference in cell growth between the 2-copy α-synuclein strain and the control Bax-containing strain was significant (P<0.05). In contrast, the difference in cell growth between 1 and 3-copy α-synuclein strains and the respective controls was insignificant (P>0.05). (C) The percentage cell death was determined by staining with phloxine B. Co-expression of 1, 2, and 3 copies of α-synuclein-HA with Bax results in much less death in cells with 2 copies of α-synuclein-HA compare to control cells which express Bax alone. There was a significant difference in cell death between the yeast strains that express different copies of α-synuclein and also their respective controls (P<0.05). (D) Microscopic images of BC300, co-expressing Bax and 1, 2, and 3 copies of α-synuclein-HA and control cells that express only Bax and contain 1, 2 and 3 copies of empty plasmids cells stained with phloxine B. (E) The amount of ROS produced in BC300, co-expressing Bax and 1, 2 and 3 copies of α-synuclein-HA was compared with the ROS produced in control cells that express only Bax and contain 1, 2, and 3 copies of empty plasmids. There is a reduction in ROS when 2 copies of HA-tagged α-synuclein was co-expressed with Bax. There was a significant difference among the three yeast strains with their respective controls (P<0.05). ANOVA test revealed that there was no significant difference in ROS levels between one and three copies (P>0.05), but there was a significant difference between one and two copies, and two and three copies (P<0.05). (F) Comparison of the MMP of BC300::Bax cells, which co-express 1, 2, and 3 copies of α-synuclein-HA and BC300::Bax cells, which co-express 1, 2, and 3 copies of empty plasmid, both controlled by GAL1 promoter. There was a significant difference between the yeast strains co-expressing Bax and 1-3 copies of α-synuclein and their respective controls (P<0.05) (G) Comparison of the levels of NDF between BC300::Bax cells, which co-express 1 copy, 2 and 3 copies of α-synuclein-HA and BC300::Bax cells, which co-express 1 copy, 2 and 3 copies of empty plasmid, via the TUNEL assay. The GAL1 promoter drove both Bax and α-synuclein gene expression. There was a significant difference in NDF in the yeast strains which co-express Bax and α-synuclein genes and which express Bax alone (P<0.05). (H) Western blots showing the presence of α-synuclein protein in cell lysates of yeast strains co-expressing Bax and one, two, or three copies of α-synuclein. 1-copy α-syn-HA (1); 2-copy α-syn-HA (2); 3-copy α-syn-HA (3), lanes 4, 5, and 6 were 1, 2, and 3 copies of an empty plasmid. 10 µg of total cellular protein was loaded in each lane. The HA-antibody was used to probe for expression of α-synuclein-HA, the c-myc antibody for Bax, and the β-actin antibody for β-actin, which was used as a loading control.