Activation of the BCL2 oncogene underlies a wide variety of malignant transformations. BCL2 must be present on the membranes of both the mitochondria and the endoplasmic reticulum (ER) to carry out its role in protecting cells from cell death (apoptosis). To enable its anchoring to intra cellular membranes, BCL2 has a C-terminal hydrophobic tail in a unique topology shared by members of a large family of Tail Anchored (TA) proteins. In mammals hundreds of such TA proteins exist that serve diverse and essential functions on every intracellular organelle. Despite the great importance of these proteins for cellular function, the pathway for enabling their anchoring to membranes has remained unknown for many years. We have recently discovered the protein machinery required for insertion of TA proteins into ER membranes in an effective and membrane specific manner in yeast. However, the mammalian pathway is not fully characterized. Moreover, the proteins required for insertion of TA proteins into mitochondrial membranes in both yeast and mammals are completely unknown. This proposal aims at uncovering and characterizing the machinery responsible for inserting BCL2 and other TA proteins into membranes in mammals. We will take two approaches – in the first, the homologous machinery to the GET complex in mammalian cells will be explored and its affect on BCL2 localization will be studied. In parallel, we will screen for components of the cellular machinery required for insertion of TA proteins into the mitochondria using high-throughput microscopy based silencing screens in mammalian cells. The discovery of the molecular machinery guiding the sub-cellular fate of the dual-localized BCL2 oncogene, can serve as a tool for better understanding BCL2 functions and for controlling its anti-apoptotic activity. More importantly, characterizing the basic machinery required for TA protein insertion will shed light on a fundamental and little explored part of cell biology.
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