Molecular Basis of the Rapamycin Insensitivity of Target Of Rapamycin Complex 2

Mol Cell. 2015 Jun 18;58(6):977-88. doi: 10.1016/j.molcel.2015.04.031. Epub 2015 May 28.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Antifungal Agents / metabolism
  • Antifungal Agents / pharmacology
  • Binding Sites / genetics
  • Biocatalysis / drug effects
  • Blotting, Western
  • Carrier Proteins / chemistry
  • Carrier Proteins / genetics
  • Carrier Proteins / metabolism
  • Cell Cycle / drug effects
  • Cell Cycle / genetics
  • Cell Cycle Proteins / chemistry
  • Cell Cycle Proteins / genetics
  • Cell Cycle Proteins / metabolism
  • Drug Resistance / genetics
  • Mass Spectrometry / methods
  • Mechanistic Target of Rapamycin Complex 2
  • Microscopy, Electron
  • Multiprotein Complexes / chemistry*
  • Multiprotein Complexes / genetics
  • Multiprotein Complexes / metabolism
  • Mutation
  • Phosphatidylinositol 3-Kinases / chemistry
  • Phosphatidylinositol 3-Kinases / genetics
  • Phosphatidylinositol 3-Kinases / metabolism
  • Protein Structure, Tertiary
  • Saccharomyces cerevisiae / drug effects
  • Saccharomyces cerevisiae / genetics
  • Saccharomyces cerevisiae / metabolism
  • Saccharomyces cerevisiae Proteins / chemistry*
  • Saccharomyces cerevisiae Proteins / genetics
  • Saccharomyces cerevisiae Proteins / metabolism
  • Saccharomyces cerevisiae Proteins / ultrastructure
  • Sirolimus / metabolism
  • Sirolimus / pharmacology*
  • TOR Serine-Threonine Kinases / chemistry*
  • TOR Serine-Threonine Kinases / genetics
  • TOR Serine-Threonine Kinases / metabolism

Substances

  • Antifungal Agents
  • Carrier Proteins
  • Cell Cycle Proteins
  • Multiprotein Complexes
  • Saccharomyces cerevisiae Proteins
  • TSC11 protein, S cerevisiae
  • TOR Serine-Threonine Kinases
  • TOR2 protein, S cerevisiae
  • Mechanistic Target of Rapamycin Complex 2
  • Sirolimus