Stoichiometry and assembly of mTOR complexes revealed by single-molecule pulldown

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. mTOR | mTORC | single molecule | stoichiometry | rapamycin

In Planta Single-Molecule Pull-Down Reveals Tetrameric Stoichiometry of HD-ZIPIII:LITTLE ZIPPER Complexes

“…The colocalization frequency of PHB-YFP and ZPR3-mCherry (;22%) is lower than values commonly reported for interacting proteins in animal systems (Jain et al, 2011(Jain et al, , 2014Giri et al, 2015). Colocalization frequency is known to be strongly influenced by fluorophore maturation probabilities; for instance, as ;80% of YFP and 40% of mCherry molecules are fluorescently active in mammalian systems, measurements of colocalization never reach 100% (reviewed in Aggarwal and Ha, 2014;Isacoff, 2007, 2008;Jain et al, 2014;Rodgers et al, 2015).…”

“…Captured proteins may be visualized with genetically encoded fluorescent tags or via immunofluorescence labeling ( Figure 1; Jain et al, 2011). SiMPull is minimally invasive, sensitive (;100-fold more sensitive than detection by immunoblotting), and rapid, allowing direct visualization of immunoprecipitated proteins at single-complex resolution in under thirty minutes (Jain et al, 2011(Jain et al, , 2014. The simplicity of this protocol also preserves weak or transient interactions that are sensitive to lengthy, invasive procedures.…”

“…The simplicity of this protocol also preserves weak or transient interactions that are sensitive to lengthy, invasive procedures. Once immobilized, colocalization analysis can be used to quantitatively assess whether coimmunoprecipitated proteins are indeed part of the same complex, and stoichiometries of these complexes can be directly determined from the stepwise pattern of fluorophore photobleaching (Jain et al, 2011(Jain et al, , 2014Yeom et al, 2011;Lee et al, 2013;Giri et al, 2015). As SiMPull is compatible with FRET (Jain et al, 2011;Krishnan et al, 2013;Hwang et al, 2014a), dynamic interactions between immunoprecipitated proteins and their binding partners can easily be tested.…”

“…As SiMPull is compatible with FRET (Jain et al, 2011;Krishnan et al, 2013;Hwang et al, 2014a), dynamic interactions between immunoprecipitated proteins and their binding partners can easily be tested. Finally, SiMPull is not limited solely to protein-protein interactions but provides single-molecule resolution data on interactions between proteins and other compounds, such as nucleic acids, small molecule ligands, and lipids (Jain et al, 2011(Jain et al, , 2014Krishnan et al, 2013;Rodgers et al, 2015;Arauz et al, 2016).…”

Defining the Domain Arrangement of the Mammalian Target of Rapamycin Complex Component Rictor Protein

“…Both mTORC1 and mTORC2 are homodimers, with a small fraction of hetero-dimer formed by both Raptor and Rictor with two mTOR ( Jain et al, 2014). The domain structure for mTOR includes a long HEAT, FAT, FRB, kinase, and FATC domains (Fig.…”