Analysis of the roles of phosphatidylinositol-4,5-bisphosphate and individual subunits in assembly, localization, and function of Saccharomyces cerevisiae target of rapamycin complex 2

Abstract

ARM, a structural motif (3-helix bundle) first described in Armadillo (Drosophila -catenin).BFP, Aequorea victoria green fluorescent protein variant carrying Y66H Y145F substitutions.CaaX box, consensus motif for protein prenylation. eGFP, Aequorea victoria green fluorescent protein variant carrying F64L S65T substitutions. EM-CCD, electron-multiplying charge-coupled device. FITC, fluoroscein isothiocyanate. 5-FOA, 5-fluoro-orotic acid. GFP, Aequorea victoria green fluorescent protein.HEAT repeat, a structural motif comprising two alpha helices connected by a short loop that is tandemly repeated in many copies to form an alpha-solenoid; named after the four proteins (Huntingtin, translation elongation factor 3, phosphoprotein phosphatase 2A, and yeast Tor1) in which this extended superhelical structure was first identified. HiLo, highly inclined and laminated optical sheet fluorescence microscopy. mAb, monoclonal antibody. MAPK, mitogen-activated protein kinase. mKate2, Entacmaea quadricolor red fluorescent protein variant. mNG, Branchiostoma lanceolatum monomeric fluorescent protein Neon Green. 1-NAA, 1-naphthaleneacetic acid (a cell-permeable synthetic auxin).ORF, open reading frame.PBS+Tw, phosphate-buffered saline containing 0.1% Tween-20 detergent.

Neomycin Interferes with Phosphatidylinositol-4,5-Bisphosphate at the Yeast Plasma Membrane and Activates the Cell Wall Integrity Pathway

“…This treatment induces PIP 2 phase separation into invaginated domains that cause TORC2 inactivation [ 49 ]. Indeed, it has been shown that PIP 2 depletion reduces TORC2 activity [ 50 ]. All these observations support the idea that neomycin is interfering with PIP 2 availability and PM organization, impairing TORC2 activity and consequently TORC2-dependent Ypk1 phosphorylation.…”

Regulation of TORC2 Function and Localization in Yeast

“…Moreover, we found that PM recruitment of Avo3 requires five or so of its more N-terminal ARM repeats (residues 496-710), which are rich in basic residues (Martinez Marshall et al 2019), suggesting that this segment of Avo3 is solvent-exposed and not closely associated with the rest of TORC2. In addition, our results indicate that docking of Avo3 on the PM is likely the initial step for de novo assembly of TORC2; once there, Avo3 attracts and tethers Tor2-Lst8 couples to the PM, which, after their dimerization, provide a scaffold to recruit the other subunits of TORC2 (Martinez Marshall et al 2019). Hence, Avo3 is essential because it has critical roles in the localization, assembly, and stability of TORC2.…”

“…It is not clear what evolutionary forces drove organisms in the fungal clade to compose TORC1 and TORC2 using two paralogous TOR proteins (Heitman et al 1991a, Shertz et al 2010, Ikai et al 2011, whereas metazoans constitute mTORC1 and mTORC2 from a single mTOR protein (Saxton & Sabatini 2017). In any event, among the distinctions between Tor1 and Tor2 observed in yeast cells early on (Kunz et al 2000, Sturgill et al 2008) and amply confirmed subsequently (Berchtold & Walther 2009, Nicastro et al 2017, Martinez Marshall et al 2019) is that Tor2 (and TORC2) is located at the plasma membrane (PM) and on endosomes, whereas Tor1 (and TORC1) is found in the cytosol and associated with the cytosolic surface of the vacuole (Figure 2). A similar distribution is observed in mammalian cells for mTORC1 (Sancak et al 2010) and mTORC2 (Ebner et al 2017).…”

“…A portion of the ARM repeat α-solenoid of Avo3 that appears to correspond to its C-terminal segment is nestled inside a cavity formed at the interface between the N-and M-HEAT repeats that mediate the main contacts between each Tor2 molecule in the dimer (Figure 3); thus, in this location, each copy of Avo3 provides additional contacts that further stabilize the Tor2 dimer situated at the heart of TORC2 (Wullschleger et al 2005, Ho et al 2008). Furthermore, our own studies have demonstrated that Avo3 is able to associate with the PM and does not rely on the presence of Tor2 or any other subunit of TORC2 to do so (Martinez Marshall et al 2019). Moreover, we found that PM recruitment of Avo3 requires five or so of its more N-terminal ARM repeats (residues 496-710), which are rich in basic residues (Martinez Marshall et al 2019), suggesting that this segment of Avo3 is solvent-exposed and not closely associated with the rest of TORC2.…”

“…Furthermore, our own studies have demonstrated that Avo3 is able to associate with the PM and does not rely on the presence of Tor2 or any other subunit of TORC2 to do so (Martinez Marshall et al 2019). Moreover, we found that PM recruitment of Avo3 requires five or so of its more N-terminal ARM repeats (residues 496-710), which are rich in basic residues (Martinez Marshall et al 2019), suggesting that this segment of Avo3 is solvent-exposed and not closely associated with the rest of TORC2. In addition, our results indicate that docking of Avo3 on the PM is likely the initial step for de novo assembly of TORC2; once there, Avo3 attracts and tethers Tor2-Lst8 couples to the PM, which, after their dimerization, provide a scaffold to recruit the other subunits of TORC2 (Martinez Marshall et al 2019).…”