homeostasis.DiscussionIn this study, we’ve systematically identified components involved in ER membrane expansion upon enforced lipid synthesis in yeast. We show that Ice2 is vital for proper ER expansion, both upon enforced lipid synthesis and during ER stress. We find that Ice2 inhibits the Nem1-Spo7 complicated, hence opposing activation of your phosphatidic acid phosphatase Pah1 and promoting membrane biogenesis. These benefits uncover an added layer of regulation of your Nem1-Spo7/Pah1 phosphatase cascade. Finally, we provide proof that Ice2 cooperates using the PA-Opi1-Ino2/4 technique to regulate ER membrane biogenesis and assists to preserve ER homeostasis. Our findings may be integrated into a model of your regulatory network that controls ER membrane biogenesis (Fig 10). At the core of this network is definitely the interconversion of DAG and PA by Dgk1 and Pah1. Ice2 inhibits Pah1 dephosphorylation by the Nem1-Spo7 complex and therefore suppresses conversion of PA into DAG. The resulting increased availability of PA is coordinated with the production of lipid synthesis enzymes that turn PA into other phospholipids. Especially, inhibition of Pah1 prevents it from repressing Ino2/4-controlled lipid synthesis genes (Santos-Rosa et al, 2005). Moreover, PA sequesters Opi1 and thereby derepresses Ino2/4 target genes (Loewen et al, 2004). Hence, inhibition of Pah1 by Ice2 increases the availability of PA and, concomitantly, induces phospholipid synthesis genes. This model readily explains the effects of ICE2 deletion and Cereblon site overexpression. Very first, the raise in LD abundance in ice2 mutants (Markgraf et al, 2014) may just result from higher constitutive Pah1 activity. The disruption of ino2-driven ER expansion by ICE2 deletion may perhaps reflect the must coordinate the production of lipid metabolic precursors with the expression of lipid synthesis genes. As we show, ino2 still induces genes encoding lipid synthesis enzymes in ice2 mutants. Nonetheless, ER expansion fails, probably because the supply of substrates for these enzymes is limiting. Precisely the same reasoning may clarify the additive effects of OPI1 deletion and ICE2 overexpression. ICE2021 The AuthorsThe EMBO Journal 40: e107958 |11 ofThe EMBO JournalDimitrios Papagiannidis et alABCDEFFigure six. Ice2 opposes Pah1 by inhibiting the Nem1-Spo7 complicated. A Schematic of Pah1 phospho-regulation. Phosphorylated Pah1 is cytosolic and inactive. Interaction of Pah1 plus the ER-localized Nem1-Spo7 complicated results in Pah1 dephosphorylation and activation, advertising conversion of PA into DAG. B CD40 Formulation Western blot of HA from WT, Dice2, Dnem1, and Dnem1 Dice2 cells expressing endogenously tagged Pah1-HA (SSY2592, 2593, 2594, 2718). Blots of SDS-PAGE and Phos-tag Web page gels are shown. C Schematic of Pah1 dephosphorylation assay with phosphorylated Pah1 from nem1 mutants and microsomes from unique strains. D Western blot of HA from Pah1 dephosphorylation reactions that contained phosphorylated Pah1-HA from nem1 mutants (SSY3065) and microsomes from cells of the indicated genotypes (SSY3053, 3074, 3075, 3095). Phosphorylated Pah1 and dephosphorylated Pah1 resulting from remedy with recombinant alkaline phosphatase (PPase) are shown for reference. E Western blot of HA, Sec61, and Pgk1 from cell lysates and microsomes prepared from WT and ice2 cells expressing Nem1-HA (SSY3140, 3141). Nem1 is undetectable in cell lysates resulting from its low abundance. F Western blot of HA from Pah1 phosphorylation reaction that contained hypophosph