Vacuolar localization via the N-terminal domain of Sch9 is required for TORC1-dependent phosphorylation and downstream signal transduction

Abstract

The budding yeast Sch9 kinase (functional ortholog of the mammalian S6 kinase) is a major effector of the Target of Rapamycin Complex 1 (TORC1) complex in the regulation of cell growth in response to nutrient availability and stress. In budding yeast, Sch9 is partially localized at the vacuolar surface, where it is phosphorylated by TORC1 under favorable growth conditions. Sch9 recruitment at the vacuole is mediated by direct interaction between PI(3,5)P2 on the vacuolar membrane and the region of Sch9 encompassing amino acid residues 1-390, which contains a C2 domain. Since many C2 domains mediate phospholipid binding, it had been suggested that the C2 domain of Sch9 mediates its vacuolar recruitment. However, the in vivo requirement of the C2 domain for Sch9 localization had not been demonstrated, and the phenotypic consequences of Sch9 delocalization remained unknown. Here, by examining cellular localization, phosphorylation state and growth phenotypes of Sch9 truncation mutants, we show that deletion of the N-terminal domain of Sch9 (aa 1-182), but not the C2 domain (aa 183-399), impairs vacuolar localization and TORC1-dependent phosphorylation of Sch9, while causing growth defects similar those observed in sch9Δ cells. Artificial tethering of an N-terminally truncated Sch9 mutant at the vacuolar membrane rescued TORC1-dependent phosphorylation and cell growth. Our study uncovers a key role for the N-terminal domain of Sch9 and demonstrates that recruitment of Sch9 at the vacuolar surface is necessary for TORC1-dependent phosphorylation and downstream signal transduction for the regulation of cell growth.

The toxic effects of yeast Ppz1 phosphatase are counteracted by subcellular relocalization mediated by its regulatory subunit Hal3

“…There are some examples of yeast proteins that translocate to the vacuolar membrane as part of their normal function. For instance, Saccharomyces cerevisiae Sch9 (a member of the AGC family of protein kinases) displays a dynamic localization, shuttling from cytosol to the vacuolar membrane where it could be phosphorylated and activated by the TORC1 complex leading to specific modulation of the Sch9 branch of TORC1 signalling [40,41]. Another example could be the PpAtg9 protein from Pichia pastoris, which localizes in structures near the plasma membrane during methanol growth and, on glucoseinduced macropexophagy, it translocates to perivacuolar structures [42].…”

Snf1/AMPK fine-tunes TORC1 signaling in response to glucose starvation

“…The Ser 288 residue lies within the C2 domain that has been suggested to mediate the vacuolar recruitment of Sch9 (Jin et al, 2014). However, recent studies have elaborated that the membrane-localization of Sch9 is primarily defined by its N-terminal 184 amino acids, but not the C2 domain (Chen et al, 2021; Novarina et al, 2021). We therefore assume that the phosphorylation of Ser 288 within the C2 domain, rather than controlling the subcellular localization, may either interfere with appropriate TORC1 docking or favor the recruitment of a pThr 737 -targeting phosphatase to Sch9.…”