Elucidating the role of ER targeted Bax and Bak and their role in inducing cell death
Creators: name: Kotevski, Aneta
Publisher: name: La Trobe
Year: 2023

description: The current dogma supports the theory that Bcl-2 associated apoptosis occurs at the mitochondria. On the other hand, it has been proposed that the pro-apoptotic Bcl-2 family member Bak can not only cause apoptosis on mitochondrial membranes, but also on the ER. In support of this, it has previously been shown that a form of Bak bearing a carboxy-terminal ER targeting sequence potently induces cell death. Based on this knowledge it poses the question of whether the mitochondria are specific targets of Bcl-2 family proteins Bax and Bak and if they are not, can Bax and Bak induce apoptosis at different locations in the cell such as the Golgi, or plasma membrane. This study endeavoured to elucidate if Bax and Bak were able to induce cell death when targeted to different membranes in the cell and if ER specific cell death was due to Bak acting directly on the ER or if the mitochondria was still involved in initiating cell death. In order to answer this question we replaced the N-terminal hydrophobic region, with a range of targeting domains. These targeting domains targeted Bax and Bak to the plasma membrane (CAAX targeting domain), trans-Golgi (GRIP targeting domain), ER (cb5 targeting domain) and the cytosol (ΔC, no targeting domain). These proteins were stably infected into bax/bak -/- fibroblasts and tested whether they would induce apoptosis Findings from this study established that neither the Golgi nor plasma membrane targeted forms of Bak nor Bax caused cell death purely based on ectopic expression. These results were contrary to Bakcb5 that showed significant cell death at a faster rate than BakWT. This suggested that Bakcb5 possessed a greater lethality compared to BakWT. Results also indicated that Bakcb5 caused a more rapid release of cytochrome c and caspase activation compared to BakWT. This suggested that a mitochondrial mediated cell death was occurring and it was hypothesised that although the cb5 targeting domain could target proteins to the ER, some misloaclisation to the mitochondria was occurring allowing Bakcb5 to be imported into the mitochondrial outer membrane. This hypothesis was confirmed by confocal imaging and in vitro import assays. Furthermore, BN-PAGE results confirmed findings from (Cheng et al., 2003) that BakWT initially forms a complex with VDAC2 that aids in the inhibition of Bak. Interestingly, Bakcb5 failed to interact with VDAC2, but oligomerised and activated at a faster rate than BakWT due to the bypassing of the VDAC2 inhibitory complex. This behaviour was further confirmed by using a positive control, BakFis as a comparison. (Lazarou et al., 2010) had shown that BakFis is imported into the OMM, bypassing the VDAC2 complex forming oligomers. This study suggests that a portion of Bakcb5 is mislocalised to the OMM and induces cell death at the mitochondria. The increased efficacy and lethality of Bakcb5 is attributed to the altered tail anchor and results in the lack of association with the VDAC2 inhibitory complex. This study has contributed new insights into understanding Bakcb5 mediated cell death and advocated the notion that Bcl-2 mediated death is mitochondrial membrane specific. Furthermore, investigation of the mouse genome via Southern and Northern blotting has led to the identification of a novel pseudogene. This pseudogene is not expressed and it was suggested that it had arisen from a viral transposition of genomic DNA. Nevertheless, this discovery is a useful source of information for the scientific community.<br><br>Submission note: A thesis submitted in total fulfilment of the requirements for the degree of Doctor of Philosophy to the Department of Biochemistry, School of Molecular Sciences, Faculty of Science, Technology and Engineering, La Trobe University, Bundoora.