To systematically explore complex genetic interactions, we constructed ~200,000 yeast triple mutants and scored negative trigenic interactions. We selected double mutant query genes across a broad spectrum of biological processes, spanning a range of quantitative features of the global digenic interaction network and tested for a genetic interaction with a third mutation. Trigenic interactions often occurred among functionally related genes and essential genes were hubs on the trigenic network. Despite their functional enrichment, trigenic interactions tended to link genes in distant bioprocesses and display a weaker magnitude than digenic interactions. We estimate that the global trigenic interaction network is ~100-fold larger than the global digenic network, highlighting the potential for complex genetic interactions to impact the biology of inheritance, including the genotype to phenotype relationship.

The target of rapamycin complex 2 (TORC2) plays a key role in maintaining the homeostasis of plasma membrane (PM) tension. TORC2 activation following increased PM tension involves redistribution of the Slm1 and 2 paralogues from PM invaginations known as eisosomes into membrane compartments containing TORC2. How Slm1/2 relocalization is triggered, and if/how this plays a role in TORC2 inactivation with decreased PM tension, is unknown. Using osmotic shocks and palmitoylcarnitine as orthogonal tools to manipulate PM tension, we demonstrate that decreased PM tension triggers spontaneous, energy-independent reorganization of pre-existing phosphatidylinositol-4,5-bisphosphate into discrete invaginated membrane domains, which cluster and inactivate TORC2. These results demonstrate that increased and decreased membrane tension are sensed through different mechanisms, highlighting a role for membrane lipid phase separation in mechanotransduction.

The Target of Rapamycin (TOR) is a eukaryotic serine/threonine protein kinase that functions in two distinct complexes, TORC1 and TORC2, to regulate growth and metabolism1,2. GTPases, responding to signals generated by abiotic stressors, nutrients, and, in metazoans, growth factors, play an important3, but poorly understood role in TORC1 regulation. Here, we report that, in budding yeast, glucose withdrawal, which leads to an acute loss of TORC1 kinase activity4, triggers a similarly rapid Rag GTPase-dependent redistribution of TORC1 from being semi-uniform around the vacuolar membrane to a single, vacuole-associated cylindrical structure visible by super-resolution optical microscopy. 3D reconstructions of cryo-electron micrograph images of these purified cylinders demonstrate that TORC1 oligomerizes into a higher-level hollow helical assembly which we name a TOROID (TORC1 Organised in Inhibited Domain). Fitting of the recently described mammalian TORC1 structure into our helical map revealed that oligomerisation leads to steric occlusion of the active site. Guided by the implications from our reconstruction, we present a TOR1 allele that prevents both TOROID formation and TORC1 inactivation in response to glucose withdrawal demonstrating that oligomerisation is necessary for TORC1 inactivation. Our results reveal a novel mechanism by which Rag-GTPases regulate TORC1 activity and suggest that the reversible assembly/disassembly of higher-level structure may be a new paradigm for the regulation of protein kinases.

SummaryMembrane contact sites (MCS) between the endoplasmic reticulum (ER) and the plasma membrane (PM) play fundamental roles in all eukaryotic cells. ER-PM MCS are particularly abundant in Saccharomyces cerevisiae, where approximately half of the PM surface is covered by cortical ER (cER). Several proteins, including Ist2, Scs2/22, and Tcb1/2/3 are implicated in cER formation, but the specific roles of these molecules are poorly understood. Here, we use cryo-electron tomography to show that ER-PM tethers are key determinants of cER morphology. Notably, Tcb proteins (tricalbins) form peaks of extreme curvature on the cER membrane facing the PM. Combined modeling and functional assays suggest that Tcb-mediated cER peaks facilitate the transport of lipids between the cER and the PM, which is necessary to maintain PM integrity under heat stress. ER peaks were also present at other MCS, implying that membrane curvature enforcement may be a widespread mechanism to regulate MCS function.