Abstract
Target of Rapamycin (TOR) plays central roles in the regulation of eukaryote growth as the hub of two essential multiprotein complexes: TORC1, which is rapamycin-sensitive, and the lesser characterized TORC2, which is not. TORC2 is a key regulator of lipid biosynthesis and Akt-mediated survival signaling. In spite of its importance, its structure and the molecular basis of its rapamycin insensitivity are unknown. Using crosslinking-mass spectrometry and electron microscopy, we determined the architecture of TORC2. TORC2 displays a rhomboid shape with pseudo-2-fold symmetry and a prominent central cavity. Our data indicate that the C-terminal part of Avo3, a subunit unique to TORC2, is close to the FKBP12-rapamycin-binding domain of Tor2. Removal of this sequence generated a FKBP12-rapamycin-sensitive TORC2 variant, which provides a powerful tool for deciphering TORC2 function in vivo. Using this variant, we demonstrate a role for TORC2 in G2/M cell-cycle progression.
Copyright © 2015 Elsevier Inc. All rights reserved.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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Antifungal Agents / metabolism
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Antifungal Agents / pharmacology
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Binding Sites / genetics
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Biocatalysis / drug effects
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Blotting, Western
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Carrier Proteins / chemistry
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Carrier Proteins / genetics
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Carrier Proteins / metabolism
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Cell Cycle / drug effects
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Cell Cycle / genetics
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Cell Cycle Proteins / chemistry
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Cell Cycle Proteins / genetics
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Cell Cycle Proteins / metabolism
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Drug Resistance / genetics
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Mass Spectrometry / methods
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Mechanistic Target of Rapamycin Complex 2
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Microscopy, Electron
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Multiprotein Complexes / chemistry*
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Multiprotein Complexes / genetics
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Multiprotein Complexes / metabolism
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Mutation
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Phosphatidylinositol 3-Kinases / chemistry
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Phosphatidylinositol 3-Kinases / genetics
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Phosphatidylinositol 3-Kinases / metabolism
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Protein Structure, Tertiary
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Saccharomyces cerevisiae / drug effects
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Saccharomyces cerevisiae / genetics
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Saccharomyces cerevisiae / metabolism
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Saccharomyces cerevisiae Proteins / chemistry*
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Saccharomyces cerevisiae Proteins / genetics
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Saccharomyces cerevisiae Proteins / metabolism
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Saccharomyces cerevisiae Proteins / ultrastructure
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Sirolimus / metabolism
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Sirolimus / pharmacology*
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TOR Serine-Threonine Kinases / chemistry*
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TOR Serine-Threonine Kinases / genetics
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TOR Serine-Threonine Kinases / metabolism
Substances
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Antifungal Agents
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Carrier Proteins
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Cell Cycle Proteins
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Multiprotein Complexes
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Saccharomyces cerevisiae Proteins
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TSC11 protein, S cerevisiae
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TOR2 protein, S cerevisiae
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Mechanistic Target of Rapamycin Complex 2
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TOR Serine-Threonine Kinases
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Sirolimus