Abstract
Saccharomyces cerevisiae target of rapamycin (TOR) complex 2 (TORC2) is an essential regulator of plasma membrane lipid and protein homeostasis. How TORC2 activity is modulated in response to changes in the status of the cell envelope is unclear. Here we document that TORC2 subunit Avo2 is a direct target of Slt2, the mitogen-activated protein kinase (MAPK) of the cell wall integrity pathway. Activation of Slt2 by overexpression of a constitutively active allele of an upstream Slt2 activator (Pkc1) or by auxin-induced degradation of a negative Slt2 regulator (Sln1) caused hyperphosphorylation of Avo2 at its MAPK phosphoacceptor sites in a Slt2-dependent manner and diminished TORC2-mediated phosphorylation of its major downstream effector, protein kinase Ypk1. Deletion of Avo2 or expression of a phosphomimetic Avo2 allele rendered cells sensitive to two stresses (myriocin treatment and elevated exogenous acetic acid) that the cell requires Ypk1 activation by TORC2 to survive. Thus, Avo2 is necessary for optimal TORC2 activity, and Slt2-mediated phosphorylation of Avo2 down-regulates TORC2 signaling. Compared with wild-type Avo2, phosphomimetic Avo2 shows significant displacement from the plasma membrane, suggesting that Slt2 inhibits TORC2 by promoting Avo2 dissociation. Our findings are the first demonstration that TORC2 function is regulated by MAPK-mediated phosphorylation.
Highlights
Cellular homeostasis requires careful coordination of metabolic growth processes and adaptive stress responses
Yeast contains two evolutionarily conserved multicomponent protein kinase complexes in which a target of rapamycin (TOR) polypeptide is the catalytic subunit: TOR complex 1 (TORC1) and TORC2
To determine whether any -SP- or -TP- sites in TORC2 subunits are direct targets of the Slt2 mitogenactivated protein kinase (MAPK), we focused initially on Avo2
Summary
Cellular homeostasis requires careful coordination of metabolic growth processes and adaptive stress responses. Ypk in turn phosphorylates multiple substrates that modulate PM lipid and protein composition (for review, see Gaubitz et al 2016; Roelants et al 2017a) It has been reported (Nomura and Inoue 2015) that TORC2 phosphorylates C-terminal turn-like and hydrophobiclike motifs in Pkc, an AGC protein kinase that controls. Other pathways that sense PM or cell wall stress may exert some of their effects via regulation of TORC2 function In this regard, two mitogenactivated protein kinase (MAPK) pathways in yeast sense and respond to stresses that challenge the cell envelope: the high-osmolarity glycerol (HOG) pathway, whose terminal MAPK is Hog (Saito and Posas 2012), and the CWI pathway, whose terminal MAPK is Slt2/Mpk (Levin 2011). Rho and its GEFs localize at sites of polarized growth to promote cell wall glucan synthesis, actin cytoskeleton organization, and targeting of secretory vesicles to growth sites (Perez and Rincón 2010)
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