Cellular homeostasis requires the ubiquitin‐dependent degradation of membrane proteins. This was assumed to be mediated exclusively either by endoplasmic reticulum‐associated degradation ( ERAD ) or by endosomal sorting complexes required for transport ( ESCRT )‐dependent lysosomal degradation. We identified in Saccharomyces cerevisiae an additional pathway that selectively extracts membrane proteins at Golgi and endosomes for degradation by cytosolic proteasomes. One endogenous substrate of this endosome and Golgi‐associated degradation pathway ( EGAD ) is the ER ‐resident membrane protein Orm2, a negative regulator of sphingolipid biosynthesis. Orm2 degradation is initiated by phosphorylation, which triggers its ER export. Once on Golgi and endosomes, Orm2 is poly‐ubiquitinated by the membrane‐embedded “Defective in SREBP cleavage” (Dsc) ubiquitin ligase complex. Cdc48/ VCP then extracts ubiquitinated Orm2 from membranes, which is tightly coupled to the proteasomal degradation of Orm2. Thereby, EGAD prevents the accumulation of Orm2 at the ER and in post‐ ER compartments and promotes the controlled de‐repression of sphingolipid biosynthesis. Thus, the selective degradation of membrane proteins by EGAD contributes to proteostasis and lipid homeostasis in eukaryotic cells.

The endosomal sorting complexes required for transport (ESCRT) mediate evolutionarily conserved membrane remodeling processes. Here, we used budding yeast (Saccharomyces cerevisiae) 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.

24 fax: +43 512 9003 73100 25 26 27 Running title: The ESCRT machinery and membrane homeostasis 28 29 2 ABSTRACT 30 31The endosomal sorting complexes required for transport (ESCRT) execute evolutionary 32 conserved membrane remodeling processes. Here we used budding yeast to explore how the 33 ESCRT machinery contributes to plasma membrane (PM) homeostasis. In response to reduced 34 membrane tension and inhibition of the target of rapamycin complex 2 (TORC2), III/Vps4 assemblies form at the PM and help to maintain membrane integrity. Conversely, the 36 growth of ESCRT mutants strongly depends on TORC2-mediated homeostatic regulation of 37 sphingolipid (SL) metabolism. This is caused by calcineurin phosphatase activity which causes 38Orm2 to accumulate at the endoplasmic reticulum (ER) in ESCRT mutants. Orm2 is a repressor 39 of SL biosynthesis and its accumulation provokes increased membrane stress. This necessitates 40 TORC2 signaling through its downstream kinase Ypk1 to control Orm2 protein levels and 41 prevent a detrimental imbalance of SL metabolism. Our findings reveal new aspects of 42 antagonistic calcineurin/TORC2 signaling for the regulation of SL biosynthesis and the 43 maintenance of PM homeostasis, and suggest that the ESCRT machinery contributes directly 44 and indirectly to these processes. 45 46 47 48 49 50 51 Key words: sphingolipid / membrane / stress / Endosomal sorting complexes required for 52 transport (ESCRT) / mTOR complex (mTORC) / calcineurin / membrane stress / TORC2 / 53 ORMDL family / Endosome and Golgi-associated degradation (EGAD) 54 Introduction 55 56 Eukaryotic cells harmonize lipid homeostasis and protein homeostasis (proteostasis) to 57 maintain the integrity of their membranes. This requires the balanced synthesis of proteins and 58 lipids and their selective degradation. How these processes are coordinated is only partially 59 understood. 60 61Selective membrane protein degradation is mediated through several ubiquitin-dependent 62 degradation pathways. In the ER, the multi-subunit transmembrane ubiquitin ligases of the ER-63 associated degradation (ERAD) machinery ubiquitinate membrane proteins for retro-64 translocation into the cytoplasm and subsequent proteasomal degradation (1,2). ERAD can also 65 degrade functional proteins in a regulated manner and thereby control membrane homeostasis. 66An important regulated ERAD substrate is the 3-hydroxy-3-methylglutaryl coenzyme A 67 reductase (HMGR), a key enzyme in sterol biosynthesis (3,4). HMGR degradation is part of a 68 rheostat that is critical for sterol homeostasis in yeast and humans. Also, squalene 69 monooxygenase is subject to sterol-regulated ERAD (5,6). When the folding and degradation 70 capacity of the ER and ERAD are overwhelmed, mis-folded proteins accumulate and induce a 71 cellular stress response pathway, called the unfolded protein response (UPR). The UPR 72 implements adaptive transcriptional programs that resolve ER stress by a decrease in overall 73 protein synthesis and a specific up-regulation of hundr...