TORC2 Signaling Is Antagonized by Protein Phosphatase 2A and the Far Complex in Saccharomyces cerevisiae

Abstract

The target of rapamycin (TOR) kinase, a central regulator of eukaryotic cell growth, exists in two essential, yet distinct, TOR kinase complexes in the budding yeast Saccharomyces cerevisiae: rapamycin-sensitive TORC1 and rapamycin-insensitive TORC2. Lst8, a component of both TOR complexes, is essential for cell viability. However, it is unclear whether the essential function of Lst8 is linked to TORC1, TORC2, or both. To that end, we carried out a genetic screen to isolate lst8 deletion suppressor mutants. Here we report that mutations in SAC7 and FAR11 suppress lethality of lst8D and TORC2-deficient (tor2-21) mutations but not TORC1 inactivation, suggesting that the essential function of Lst8 is linked only to TORC2. More importantly, characterization of lst8D bypass mutants reveals a role for protein phosphatase 2A (PP2A) in the regulation of TORC2 signaling. We show that Far11, a member of the Far3-7-8-9-10-11 complex involved in pheromone-induced cell cycle arrest, interacts with Tpd3 and Pph21, conserved components of PP2A, and deletions of components of the Far3-7-8-9-10-11 complex and PP2A rescue growth defects in lst8D and tor2-21 mutants. In addition, loss of the regulatory B9 subunit of PP2A Rts1 or Far11 restores phosphorylation to the TORC2 substrate Slm1 in a tor2-21 mutant. Mammalian Far11 orthologs FAM40A/B exist in a complex with PP2A known as STRIPAK, suggesting a conserved functional association of PP2A and Far11. Antagonism of TORC2 signaling by PP2A-Far11 represents a novel regulatory mechanism for controlling spatial cell growth of yeast.T HE target of rapamycin (TOR) kinase is a phosphatidylinositol kinase-related protein kinase that controls eukaryotic cell growth and proliferation in response to nutrient conditions (Inoki et al. 2005;Wullschleger et al. 2006;Zoncu et al. 2011). The TOR kinase is inhibited by the complex of rapamycin and Fpr1, a peptidyl-prolyl cis-trans isomerase. The TOR kinase is conserved in eukaryotes. Unlike fungal species, which may possess two TOR kinases, higher eukaryotes such as humans possess only one. The TOR kinase exists in multi-protein complexes, which have been purified from many different eukaryotic systems. There exist two distinct TOR kinase complexes. In yeast, rapamycin-sensitive TORC1 consists of Tor1 or Tor2, Lst8, Kog1, and Tco89, and rapamycin-insensitive TORC2 consists of Tor2, Lst8, Avo1, Avo2, Avo3, and Bit61 (Loewith et al. 2002;Wedaman et al. 2003;Reinke et al. 2004). Both complexes are partially conserved in mammals: mTORC1 contains the yeast Kog1 ortholog raptor, while mTORC2 contains mSin1 and rictor, orthologs of yeast Avo1 and Avo3, respectively; GbL, the ortholog of yeast Lst8, exists in both mTORC1 and mTORC2 (Zoncu et al. 2011).TOR regulates cell growth by sensing and responding to changes in nutrient conditions (Schmelzle and Hall 2000). TORC1 has an essential function involving the regulation of cell growth that is carried out when either Tor1 or Tor2 is in the complex. Under favorable growth conditions, TORC1 promotes ce...

Tiered Assembly of the Yeast Far3-7-8-9-10-11 Complex at the Endoplasmic Reticulum

“…7B), indicating that the tail-anchor domain of Far9 is required for its ER localization. Recently, we proposed that the Far complex antagonizes TORC2 signaling by showing that a far9⌬ mutation or loss of other Far complex components are able to bypass a tor2 temperature-sensitive (tor2-21) mutation (24). We sought to test whether ER localization of the Far complex is required for its function.…”

“…In the budding yeast Saccharomyces cerevisiae, the STRIPAK complex has been reported to mediate pheromone signaling, the TORC2 signaling pathway, and the toxicity due to expression of human caspase-10 in yeast (21)(22)(23)(24). The yeast STRIPAK complex contains Far3, Far7, Far8, Far9, Far10, Far11, Tpd3 (the A scaffolding subunit of PP2A), and Pph21/22 (the two redundant catalytic subunits of PP2A) (22)(23)(24)(25)(26).…”

“…The yeast STRIPAK complex contains Far3, Far7, Far8, Far9, Far10, Far11, Tpd3 (the A scaffolding subunit of PP2A), and Pph21/22 (the two redundant catalytic subunits of PP2A) (22)(23)(24)(25)(26). Far11 is an ortholog of human STRIP1/2; Far8 shares limited sequence similarity with human striatins; Far9 and Far10 are homologous tail-anchored proteins similar to human SLMAP (Table 1) (1,27).…”

“…Mutations in FAR genes lead to increased resistance to pheromoneinduced cell cycle arrest (22,29), but the underlying mechanism is still unclear. The targets of rapamycin kinases are conserved in eukaryotes and exist in two distinct multiprotein complexes, TORC1 and TORC2 (30,31), and mutations in the yeast STRIPAK complex components lead to suppression of cell lethality specifically due to TORC2 deficiency possibly by restoring phosphorylation of TORC2 substrates Slm1, Slm2, Ypk1, and Ypk2 (21,24,32,33). The role of STRIPAK in human caspase-10-induced toxicity in yeast likely results from promoting Atg13 dephosphorylation and subsequent activation of autophagy (23,34).…”

“…In yeast, Far3, Far7, Far8, Far9, Far10, and Far11 have been reported to form a complex (22,24,25). However, it is unclear how these proteins assemble together to form the final complex, and identification of the cellular component of this complex could potentially provide insights into the mechanism of its function.…”