TOR complex 2 (TORC2) signaling and the ESCRT machinery cooperate in the protection of plasma membrane integrity in yeast

Abstract

<jats:p>The endosomal sorting complexes required for transport (ESCRT) mediate evolutionarily conserved membrane remodeling processes. Here, we used budding yeast (<jats:italic>Saccharomyces cerevisiae</jats:italic>) to explore how the ESCRT machinery contributes to plasma membrane (PM) homeostasis. We found that in response to reduced membrane tension and inhibition of TOR complex 2 (TORC2), ESCRT-III/Vps4 assemblies form at the PM and help maintain membrane integrity. In turn, the growth of ESCRT mutants strongly depended on TORC2-mediated homeostatic regulation of sphingolipid (SL) metabolism. This was caused by calcineurin-dependent dephosphorylation of Orm2, a repressor of SL biosynthesis. Calcineurin activity impaired Orm2 export from the endoplasmic reticulum (ER) and thereby hampered its subsequent endosome and Golgi-associated degradation (EGAD). The ensuing accumulation of Orm2 at the ER in ESCRT mutants necessitated TORC2 signaling through its downstream kinase Ypk1, which repressed Orm2 and prevented a detrimental imbalance of SL metabolism. Our findings reveal compensatory cross-talk between the ESCRT machinery, calcineurin/TORC2 signaling, and the EGAD pathway important for the regulation of SL biosynthesis and the maintenance of PM homeostasis.</jats:p>

Regulation of sphingolipid biosynthesis in the endoplasmic reticulum via signals from the plasma membrane in budding yeast

“…We predicted that under dysfunction of ceramide synthesis, inactivation due to excessive phosphorylation of Orm1 and may also Orm2, which might be a more potent SPT inhibitor [18,35], could lead to overactivation of SPT, which in turn leads to an increase in intracellular LCBs and LCBPs, thereby causing cytotoxicity [36,37]. Therefore, it is expected that cells might prevent excessive phosphorylation of Orm1/2 through Ypk1 somehow to avoid such events, possibly through dephosphorylation of Orm1/ 2.…”

Plasma membrane damage limits replicative lifespan in yeast and induces premature senescence in human fibroblasts

“…ESCRT can contribute to the replicative lifespan by at least two scenarios that are not mutually exclusive. First, ESCRT-dependent plasma membrane repair may prevent cell lysis and extend the replicative lifespan through the ESCRT’s function in plasma membrane repair as established in human cells (11, 26) and recently suggested in yeast (27). Second, ESCRT-dependent vacuolar membrane repair may extend the replicative lifespan of budding yeast.…”

Membrane Contact Sites in Yeast: Control Hubs of Sphingolipid Homeostasis

“…Simultaneously, heat stress induces Ca 2+ influx and Ca 2+ /calmodulinmediated activation of the protein phosphatase calcineurin, which reverses Lag1 and Lac1 phosphorylation [58,59]. In addition, calcineurin activates ORM2 transcription [8] and represses Orm1/2 degradation via the EGAD pathway by promoting dephosphorylation of Orm1/2 either directly or indirectly [101]. The opposite roles of TORC2 and calcineurin were further demonstrated by the fact that deletion of the calcineurin regulatory subunit B restored phytoceramide levels in cells containing a temperature-sensitive allele of the TORC2 subunit TSC3 [59].…”

“…Later studies reported that the ESCRT machinery is employed for membrane remodeling at other sides as well [110]. Schmidt et al showed that the ESCRT III complex and Vps4 are recruited to the PM to maintain membrane integrity, in response to reduced membrane tension and TORC2 inhibition [101]. Conversely, deletion of Vps4 causes hypersensitivity to PM stress, deregulated calcineurin signaling, and activation of TORC2 signaling as a counter measure to calcineurin-inhibited sphingolipid synthesis [101].…”

“…Schmidt et al showed that the ESCRT III complex and Vps4 are recruited to the PM to maintain membrane integrity, in response to reduced membrane tension and TORC2 inhibition [101]. Conversely, deletion of Vps4 causes hypersensitivity to PM stress, deregulated calcineurin signaling, and activation of TORC2 signaling as a counter measure to calcineurin-inhibited sphingolipid synthesis [101]. Therefore, such as ER-PM contact sites, ESCRT III/Vps4 acts as a mediator of the TORC2/Ca 2+ rheostat and possibly provides a back-up mechanism when ER-PM contacts are deleted.…”