Phosphorylation by the stress-activated MAPK Slt2 down-regulates the yeast TOR complex 2

The TOR complex 2 (TORC2) in yeast is known to regulate plasma membrane lipid and protein homeostasis. However, how TORC2 activity is modulated in response to cell envelope stress remains unclear. Prior research has shown TORC2's role in maintaining membrane integrity, but the specific mechanisms linking stress signaling to TORC2 remain unexplored. This gap motivated the investigation into how stress-activated MAPKs might influence TORC2. No prior work had resolved whether MAPKs like Slt2 could directly modulate TORC2 activity. Existing studies suggest TORC2 interacts with downstream effectors like Ypk1, but the regulatory mechanisms remain unclear. This uncertainty drove the need to examine TORC2's response to MAPK signaling. Understanding these interactions could clarify how yeast cells adapt to environmental stressors. This paper addresses that need by focusing on Slt2's role in TORC2 regulation.

This study aimed to determine whether the stress-activated MAPK Slt2 modulates TORC2 activity in yeast. The researchers focused on the TORC2 subunit Avo2 as a potential target of Slt2. Their goal was to investigate how Slt2-mediated phosphorylation affects TORC2 signaling. They sought to establish whether Slt2 activity influences TORC2's ability to phosphorylate Ypk1. The study also aimed to assess the consequences of Avo2 phosphorylation on TORC2 function. They tested whether Avo2 is essential for TORC2 activity and how its phosphorylation impacts cell survival under stress. The motivation was to clarify the regulatory mechanisms linking stress signaling to TORC2. This work contributes to understanding how yeast cells respond to envelope stress.

The researchers used genetic and biochemical approaches to study TORC2 regulation. They activated Slt2 by overexpressing a constitutively active Pkc1 allele or degrading Sln1 with auxin. Phosphorylation of Avo2 was assessed using phospho-specific antibodies. They examined Ypk1 phosphorylation as a downstream TORC2 effector. Deletion mutants and phosphomimetic Avo2 alleles were created to test TORC2 function. Cell survival was tested under stress conditions like myriocin and acetic acid. Localization of Avo2 at the plasma membrane was analyzed using fluorescence microscopy. These methods allowed the researchers to link Slt2 activity to TORC2 signaling.

Slt2 activation led to hyperphosphorylation of Avo2 at MAPK phosphoacceptor sites. This phosphorylation occurred in a Slt2-dependent manner and reduced TORC2-mediated Ypk1 phosphorylation. Deletion of Avo2 or phosphomimetic Avo2 expression caused sensitivity to myriocin and acetic acid. These stresses require TORC2/Ypk1 signaling for cell survival. Phosphomimetic Avo2 showed significant displacement from the plasma membrane. This suggests Slt2 inhibits TORC2 by promoting Avo2 dissociation. TORC2 activity was diminished under Slt2 activation conditions. The findings indicate Slt2-mediated phosphorylation down-regulates TORC2 signaling.

The authors propose that Slt2-mediated phosphorylation of Avo2 down-regulates TORC2 signaling. Their findings suggest Avo2 is necessary for optimal TORC2 activity. Phosphomimetic Avo2 displacement from the plasma membrane supports this conclusion. The study shows TORC2 function is regulated by MAPK-mediated phosphorylation. Slt2 activation reduces TORC2's ability to phosphorylate Ypk1. This effect is observed under stress conditions like myriocin and acetic acid. The results imply TORC2 signaling is modulated in response to cell envelope stress. These conclusions are based on the observed effects of Avo2 phosphorylation and Ypk1 activity.