Stoichiometry and Assembly of mTOR Complexes Revealed by Single-Molecule Pulldown

Proc Natl Acad Sci U S A. 2014 Dec 16;111(50):17833-8. doi: 10.1073/pnas.1419425111. Epub 2014 Dec 1.

Abstract

The mammalian target of rapamycin (mTOR) kinase is a master regulator of cellular, developmental, and metabolic processes. Deregulation of mTOR signaling is implicated in numerous human diseases including cancer and diabetes. mTOR functions as part of either of the two multisubunit complexes, mTORC1 and mTORC2, but molecular details about the assembly and oligomerization of mTORCs are currently lacking. We use the single-molecule pulldown (SiMPull) assay that combines principles of conventional pulldown assays with single-molecule fluorescence microscopy to investigate the stoichiometry and assembly of mTORCs. After validating our approach with mTORC1, confirming a dimeric assembly as previously reported, we show that all major components of mTORC2 exist in two copies per complex, indicating that mTORC2 assembles as a homodimer. Interestingly, each mTORC component, when free from the complexes, is present as a monomer and no single subunit serves as the dimerizing component. Instead, our data suggest that dimerization of mTORCs is the result of multiple subunits forming a composite surface. SiMPull also allowed us to distinguish complex disassembly from stoichiometry changes. Physiological conditions that abrogate mTOR signaling such as nutrient deprivation or energy stress did not alter the stoichiometry of mTORCs. On the other hand, rapamycin treatment leads to transient appearance of monomeric mTORC1 before complete disruption of the mTOR-raptor interaction, whereas mTORC2 stoichiometry is unaffected. These insights into assembly of mTORCs may guide future mechanistic studies and exploration of therapeutic potential.

Keywords: mTOR; mTORC; rapamycin; single molecule; stoichiometry.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Bacterial Proteins
  • Blotting, Western
  • Dimerization
  • HEK293 Cells
  • Humans
  • Immunoprecipitation
  • Luminescent Proteins
  • Mechanistic Target of Rapamycin Complex 1
  • Mechanistic Target of Rapamycin Complex 2
  • Microscopy, Fluorescence
  • Models, Molecular
  • Multiprotein Complexes / chemistry*
  • Multiprotein Complexes / metabolism
  • Sirolimus / pharmacology
  • TOR Serine-Threonine Kinases / chemistry*
  • TOR Serine-Threonine Kinases / metabolism

Substances

  • Bacterial Proteins
  • Luminescent Proteins
  • Multiprotein Complexes
  • red fluorescent protein
  • yellow fluorescent protein, Bacteria
  • TOR Serine-Threonine Kinases
  • Mechanistic Target of Rapamycin Complex 1
  • Mechanistic Target of Rapamycin Complex 2
  • Sirolimus