title: Molecular Basis of the Rapamycin Insensitivity of Target Of Rapamycin Complex 2.
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.
authors.affiliation: Department of Molecular Biology and Institute of Genetics and Genomics of Geneva (iGE3), University of Geneva, 30 Quai Ernest Ansermet, CH1211 Geneva, Switzerland.
authors.forename: Christl
authors.initials: C
authors.lastname: Gaubitz
authors.affiliation: European Molecular Biology Laboratory, Grenoble Outstation, 71 Avenue des Martyrs, 38042 Grenoble, France; Fondation ARC, 9 rue Guy Môquet, BP 90003, 04803 Villejuif Cedex, France.
authors.forename: Taiana M
authors.initials: TM
authors.lastname: Oliveira
authors.affiliation: Department of Molecular Biology and Institute of Genetics and Genomics of Geneva (iGE3), University of Geneva, 30 Quai Ernest Ansermet, CH1211 Geneva, Switzerland.
authors.forename: Manoel
authors.initials: M
authors.lastname: Prouteau
authors.affiliation: Department of Biology, Institute of Molecular Systems Biology, ETH Zürich, 8093 Zürich, Switzerland.
authors.forename: Alexander
authors.initials: A
authors.lastname: Leitner
authors.affiliation: European Molecular Biology Laboratory, Grenoble Outstation, 71 Avenue des Martyrs, 38042 Grenoble, France.
authors.forename: Manikandan
authors.initials: M
authors.lastname: Karuppasamy
authors.affiliation: Department of Molecular Biology and Institute of Genetics and Genomics of Geneva (iGE3), University of Geneva, 30 Quai Ernest Ansermet, CH1211 Geneva, Switzerland.
authors.forename: Georgia
authors.initials: G
authors.lastname: Konstantinidou
authors.affiliation: Department of Molecular Biology and Institute of Genetics and Genomics of Geneva (iGE3), University of Geneva, 30 Quai Ernest Ansermet, CH1211 Geneva, Switzerland.
authors.forename: Delphine
authors.initials: D
authors.lastname: Rispal
authors.affiliation: Department of Molecular Biology and Institute of Genetics and Genomics of Geneva (iGE3), University of Geneva, 30 Quai Ernest Ansermet, CH1211 Geneva, Switzerland.
authors.forename: Sandra
authors.initials: S
authors.lastname: Eltschinger
authors.affiliation: Department of Molecular Biology and Institute of Genetics and Genomics of Geneva (iGE3), University of Geneva, 30 Quai Ernest Ansermet, CH1211 Geneva, Switzerland.
authors.forename: Graham C
authors.initials: GC
authors.lastname: Robinson
authors.affiliation: Department of Molecular Biology and Institute of Genetics and Genomics of Geneva (iGE3), University of Geneva, 30 Quai Ernest Ansermet, CH1211 Geneva, Switzerland; University of Bordeaux, European Institute for Chemistry and Biology, ARNA Laboratory, F-33607 Pessac, France; Institut National de la Santé Et de la Recherche Médicale, INSERM-U869, ARNA Laboratory, F-33000, Bordeaux, France.
authors.forename: Stéphane
authors.initials: S
authors.lastname: Thore
authors.affiliation: Department of Biology, Institute of Molecular Systems Biology, ETH Zürich, 8093 Zürich, Switzerland; Faculty of Science, University of Zürich, 8057 Zürich, Switzerland.
authors.forename: Ruedi
authors.initials: R
authors.lastname: Aebersold
authors.affiliation: European Molecular Biology Laboratory, Grenoble Outstation, 71 Avenue des Martyrs, 38042 Grenoble, France; School of Biochemistry, University of Bristol, Bristol, BS8 1TD, United Kingdom. Electronic address: schaffitzel@embl.fr.
authors.forename: Christiane
authors.initials: C
authors.lastname: Schaffitzel
authors.affiliation: Department of Molecular Biology and Institute of Genetics and Genomics of Geneva (iGE3), University of Geneva, 30 Quai Ernest Ansermet, CH1211 Geneva, Switzerland; National Centre of Competence in Research "Chemical Biology," University of Geneva, Geneva CH-1211, Switzerland. Electronic address: robbie.loewith@unige.ch.
authors.forename: Robbie
authors.initials: R
authors.lastname: Loewith
mesh_terms: D000935:Antifungal Agents / Q000378:metabolism / Q000494:pharmacology; D001665:Binding Sites / Q000235:genetics; D055162:Biocatalysis / Q000187:drug effects; D015153:Blotting, Western; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D002453:Cell Cycle / Q000187:drug effects / Q000235:genetics; D018797:Cell Cycle Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004351:Drug Resistance / Q000235:genetics; D013058:Mass Spectrometry / Q000379:methods; D000076225:Mechanistic Target of Rapamycin Complex 2; D008854:Microscopy, Electron; D046912:Multiprotein Complexes / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D009154:Mutation; D019869:Phosphatidylinositol 3-Kinases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017434:Protein Structure, Tertiary; D012441:Saccharomyces cerevisiae / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism / Q000648:ultrastructure; D020123:Sirolimus / Q000378:metabolism / Q000494:pharmacology*; D058570:TOR Serine-Threonine Kinases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
chemical_list: D000935:Antifungal Agents; D002352:Carrier Proteins; D018797:Cell Cycle Proteins; D000076225:Mechanistic Target of Rapamycin Complex 2; D046912:Multiprotein Complexes; D019869:Phosphatidylinositol 3-Kinases; D029701:Saccharomyces cerevisiae Proteins; D020123:Sirolimus; D058570:TOR Serine-Threonine Kinases; C504817:TSC11 protein, S cerevisiae; C081135:TOR2 protein, S cerevisiae