BCL-2 proteins are tail-anchored (TA) proteins localized to the mitochondrial outer membrane (MOM). Although their roles in regulating apoptosis are well understood, how they are inserted into the mitochondria remains to be characterized. As many TA proteins are inserted by an ASNA1-mediated pathway, here I examined if ASNA1 is involved in BCL-2 protein insertion. Furthermore, despite the pivotal role of ASNA1 in regulating many cellular functions, the essentiality of ASNA1 in cancer and normal cells is still to be fully addressed. Using a conditional knockout system, I discovered that ASNA1 was essential for the survival of HeLa and RPE-1 cells. MCL-1, an anti-apoptotic member of the BCL-2 family, was found to interact with ASNA1 using co-immunoprecipitation assays. Moreover, a positiv...[ Read more ]

BCL-2 proteins are tail-anchored (TA) proteins localized to the mitochondrial outer membrane (MOM). Although their roles in regulating apoptosis are well understood, how they are inserted into the mitochondria remains to be characterized. As many TA proteins are inserted by an ASNA1-mediated pathway, here I examined if ASNA1 is involved in BCL-2 protein insertion. Furthermore, despite the pivotal role of ASNA1 in regulating many cellular functions, the essentiality of ASNA1 in cancer and normal cells is still to be fully addressed. Using a conditional knockout system, I discovered that ASNA1 was essential for the survival of HeLa and RPE-1 cells. MCL-1, an anti-apoptotic member of the BCL-2 family, was found to interact with ASNA1 using co-immunoprecipitation assays. Moreover, a positive correlation in protein expression was identified between MCL-1 and ASNA1. Depletion of ASNA1 downregulated the expression of MCL-1. Conversely, overexpression of ASNA1 stabilized MCL-1 protein. Moreover, knockout of ASNA1 sensitized cells to a MCL-1 inhibitor, which suggests a potential synergism between ASNA1 and MCL-1 in inducing cell death. In addition to ASNA1, the essentiality of BCL-2 and BCL-XL in cancer cells was also addressed using the condition knockout system. BCL-XL, but not BCL-2, was essential for cell survival in unperturbed and mitotic-arrested HeLa cells. Knockout of BCL-XL promoted cell death in a BAX-dependent manner. Surprisingly, co-depletion of BCL-2 and BCL-XL synergistically induced cell death, implicating their functional redundancy in cell survival. Altogether, these results extend our understanding of the regulation of BCL-2 proteins and provide insights into therapeutics development for targeting BCL-2 proteins.