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

Abstract

Eukaryotic cell survival requires maintenance of plasma membrane (PM) homeostasis in response to environmental insults and changes in lipid metabolism. In yeast, a key regulator of PM homeostasis is target of rapamycin (TOR) complex 2 (TORC2), a multiprotein complex containing the evolutionarily conserved TOR protein kinase isoform Tor2. PM localization is essential for TORC2 function. One core TORC2 subunit (Avo1) and two TORC2-­associated regulators (Slm1 and Slm2) contain pleckstrin homology (PH) domains that exhibit specificity for binding phosphatidylinositol-4,5- bisphosphate (PtdIns4,5P2). To investigate the roles of PtdIns4,5P2 and constituent subunits of TORC2, we used auxin-inducible degradation to systematically eliminate these factors and then examined localization, association, and function of the remaining TORC2 components. We found that PtdIns4,5P2 depletion significantly reduced TORC2 activity, yet did not prevent PM localization or cause disassembly of TORC2. Moreover, truncated Avo1 (lacking its C-terminal PH domain) was still recruited to the PM and supported growth. Even when all three PH-containing proteins were absent, the remaining TORC2 subunits were PM-bound. Revealingly, Avo3 localized to the PM independent of both Avo1 and Tor2, whereas both Tor2 and Avo1 required Avo3 for their PM anchoring. Our findings provide new mechanistic information about TORC2 and pinpoint Avo3 as pivotal for TORC2 PM localization and assembly in vivo.

The flipside of the TOR coin – TORC2 and plasma membrane homeostasis at a glance

“…Avo3 has an important scaffolding function since its depletion triggers the disassembly of TORC2 (Wullschleger et al, 2005). It also contributes to the PM localization of TORC2 (Martinez Marshall et al, 2019). Avo1 possesses a PH domain that is able to bind various phosphoinositides and has been reported to tether TORC2 to the PM (Berchtold and Walther, 2009).…”

“…Based on the observation that deletion of the PH domain of Avo1 is lethal, but can be rescued by its replacement with another PMtargeting domain (the CAAX box), Avo1 has been generally assumed to mediate the anchoring of TORC2 to the PM though an interaction with PI(4,5)P 2 (Berchtold and Walther, 2009). However, a recent study proposes a different model, in which the N-terminal armadillo repeats of Avo3 anchor TORC2 to the PM independently of PI(4,5)P 2 , either through a direct interaction with negatively charged head groups of PM phospholipids, or indirectly through a yet unidentified PM protein (Martinez-Marshall et al, 2019). This work confirmed the importance of PI(4,5)P 2 for TORC2 activity, possibly through eisosome formation and/or the recruitment of Slm1 and/or Slm2 to the PM (Martinez-Mashall et al, 2019).…”

RTKN-1/Rhotekin shields endosome-associated F-actin from disassembly to ensure endocytic recycling

“…S2, B and B'). PH domain often exhibits an affinity for PI(4,5)P2 (Martinez Marshall et al, 2019). However, the RTKN-1-PH domain solely resided in punctate structures and partially overlapped with Tubby-PH (R332H) -mCherry (Fig.…”

Regulation of TORC2 function and localization by Rab5 GTPases inSaccharomyces cerevisiae

“…Basal activity and stability of Ypk1 also requires its phosphorylation at S644, which lies within a conserved sequence (dubbed the "turn motif") located downstream of its kinase homology domain within a C-terminal regulatory domain [16]. This modification is installed by TORC2, which is also largely PM-associated [17][18][19][20][21]. Under certain stressful conditions that stimulate TORC2-mediated phosphorylation of Ypk1, such as sphingolipid limitation [22], heat stress [23], hypotonic conditions [24,25], and acetic acid stress [26], TORC2 further elevates Ypk1 activity by phosphorylating four additional sites in its C-terminal regulatory domain, paramount among them is T662, which lies within another conserved sequence (dubbed the "hydrophobic motif") in the C-terminal domain [13,16].…”

“…Where? Localization of TORC2 in S. cerevisiae to discrete puncta residing at the cell cortex has been well documented, based on analysis of fixed cells by indirect immunofluorescence [20] or immunogold EM [6], or examination of live cells expressing fluorescently-tagged derivatives of Tor2 [18,19,21] or TORC2-specific subunits that associate tightly with Tor2 [17,19,21]. In addition, the effector protein kinases (Ypk1 and Ypk2) directly phosphorylated by TORC2 are, as mentioned earlier, also obligatorily phosphorylated by other protein kinases (Pkh1 and Pkh2) that are tightly associated with the PM-anchored eisosomes.…”