Detection of Cytoplasmic and Nuclear Functions of mTOR by Fractionation

摘要: Subcellular localization constitutes the environment in which proteins act. It tightly controls access to and availability of different types molecular interacting partners is therefore a major determinant protein function regulation. Originally thought be mere cytoplasmic kinase mammalian target rapamycin (mTOR) has recently been localized various intracellular compartments including nucleus specific components endomembrane system such as lysosomes. The identification essential binding structural functional partitioning mTOR into two distinct multiprotein complexes warrant detailed investigation subcellular part mTORC1 mTORC2. Upon establishment experimental conditions allowing cytoplasmic/nuclear fractionation at high purity maximum complex recovery we have previously shown that mTOR/raptor (mTORC1) predominantly whereas mTOR/rictor (mTORC2) abundant both compartments. Moreover, mTORC2 rictor sin1 are dephosphorylated dynamically distributed between cytoplasm upon long-term treatment with mTOR-inhibitor rapamycin. These findings further demonstrate here presented detailly described procedure valuable tool study shuttling context expanding signalling.