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Open Access Publications from the University of California

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.

  • Author(s): Martinez Marshall, Maria Nieves
  • Emmerstorfer-Augustin, Anita
  • Leskoske, Kristin L
  • Zhang, Lydia H
  • Li, Biyun
  • Thorner, Jeremy
  • et al.

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 multi-protein 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 dissassembly 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 independently 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.

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