biostudies-literatureRoelants FMBiotechnology and Biological Sciences Research CouncilNIGMS NIH HHS2128-2136https://www.ebi.ac.uk/biostudies/studies/S-EPMC6232965biostudies-literatureUnknown29(17)In our proteome-wide screen, Ysp2 (also known as Lam2/Ltc4) was identified as a likely physiologically relevant target of the TOR complex 2 (TORC2)-dependent protein kinase Ypk1 in the yeast Saccharomyces cerevisiae. Ysp2 was subsequently shown to be one of a new family of sterol-binding proteins located at plasma membrane (PM)-endoplasmic reticulum (ER) contact sites. Here we document that Ysp2 and its paralogue Lam4/Ltc3 are authentic Ypk1 substrates in vivo and show using genetic and biochemical criteria that Ypk1-mediated phosphorylation inhibits the ability of these proteins to promote retrograde transport of sterols from the PM to the ER. Furthermore, we provide evidence that a change in PM sterol homeostasis promotes cell survival under membrane-perturbing conditions known to activate TORC2-Ypk1 signaling. These observations define the underlying molecular basis of a new regulatory mechanism for cellular response to plasma membrane stress.Molecular biology of the cellTOR complex 2-regulated protein kinase Ypk1 controls sterol distribution by inhibiting StARkin domain-containing proteins located at plasma membrane-endoplasmic reticulum contact sites.PMC6232965R01 GM021841BB/P003818/1BB/P003818Davis JCChauhan NMuir AMenon AKThorner JRoelants FMLevine TPfalseTOR complex 2-regulated protein kinase Ypk1 controls sterol distribution by inhibiting StARkin domain-containing proteins located at plasma membrane-endoplasmic reticulum contact sites.In our proteome-wide screen, Ysp2 (also known as Lam2/Ltc4) was identified as a likely physiologically relevant target of the TOR complex 2 (TORC2)-dependent protein kinase Ypk1 in the yeast Saccharomyces cerevisiae. Ysp2 was subsequently shown to be one of a new family of sterol-binding proteins located at plasma membrane (PM)-endoplasmic reticulum (ER) contact sites. Here we document that Ysp2 and its paralogue Lam4/Ltc3 are authentic Ypk1 substrates in vivo and show using genetic and biochemical criteria that Ypk1-mediated phosphorylation inhibits the ability of these proteins to promote retrograde transport of sterols from the PM to the ER. Furthermore, we provide evidence that a change in PM sterol homeostasis promotes cell survival under membrane-perturbing conditions known to activate TORC2-Ypk1 signaling. These observations define the underlying molecular basis of a new regulatory mechanism for cellular response to plasma membrane stress.2018-01-01T00:00:00Z2018 Aug2022-02-09T10:23:54.245Z2019-03-27T00:08:25ZS-EPMC62329652992735110.1091/mbc.E18-04-0229