title: Detection of cytoplasmic and nuclear functions of mTOR by fractionation.
abstract: Subcellular localization constitutes the environment in which proteins act. It tightly controls access to and availability of different types of molecular interacting partners and is therefore a major determinant of protein function and regulation. Originally thought to be a mere cytoplasmic kinase the mammalian target of rapamycin (mTOR) has recently been localized to various intracellular compartments including the nucleus and specific components of the endomembrane system such as lysosomes. The identification of essential binding partners and the structural and functional partitioning of mTOR into two distinct multiprotein complexes warrant the detailed investigation of the subcellular localization of mTOR as part of mTORC1 and mTORC2. Upon establishment of experimental conditions allowing cytoplasmic/nuclear fractionation at high purity and maximum mTOR complex recovery we have previously shown that the mTOR/raptor complex (mTORC1) is predominantly cytoplasmic whereas the mTOR/rictor complex (mTORC2) is abundant in both compartments. Moreover, the mTORC2 complex components rictor and sin1 are dephosphorylated and dynamically distributed between the cytoplasm and the nucleus upon long-term treatment with the mTOR-inhibitor rapamycin. These findings further demonstrate that the here presented and detailly described fractionation procedure is a valuable tool to study protein localization and cytoplasmic/nuclear protein shuttling in the context of expanding mTOR signalling.
authors: Medical Genetics, Medical University of Vienna, Vienna, Austria.
authors: Margit
authors: M
authors: Rosner
authors: Markus
authors: M
authors: Hengstschläger
mesh_terms: D000818:Animals; D002458:Cell Fractionation / Q000379:methods*; D002467:Cell Nucleus / Q000378:metabolism*; D003593:Cytoplasm / Q000378:metabolism*; D003600:Cytosol; D005347:Fibroblasts / Q000378:metabolism; D005822:Genetic Vectors; D006801:Humans; D000076222:Mechanistic Target of Rapamycin Complex 1; D046912:Multiprotein Complexes / Q000378:metabolism*; D010766:Phosphorylation; D011506:Proteins / Q000378:metabolism; D015398:Signal Transduction; D058570:TOR Serine-Threonine Kinases; D014157:Transcription Factors / Q000378:metabolism
chemical_list: C493245:CRTC2 protein, human; D046912:Multiprotein Complexes; D011506:Proteins; D014157:Transcription Factors; D000076222:Mechanistic Target of Rapamycin Complex 1; D058570:TOR Serine-Threonine Kinases