Rewiring phospholipid biosynthesis reveals robustness in membrane homeostasis and uncovers lipid regulatory players

Abstract

Intracellular transport of lipids by Lipid Transport Proteins (LTPs) is thought to work alongside vesicular transport to shuttle lipids from their place of synthesis to their destinations. Whereas many LTPs have been identified, it is largely unknown which routes and which LTPs a given lipid utilizes to navigate the multiple membranes of eukaryotic cells. The major and essential phospholipids, phosphatidylethanolamine (PE) and phosphatidylcholine (PC) can be produced by multiple pathways and, in the case of PE, also at multiple locations. Here, we present an approach in which we simplify and rewire yeast phospholipid synthesis by redirecting PE and PC synthesis reactions to distinct subcellular locations using chimeric enzymes fused to specific organelle targeting motifs. In rewired conditions, viability is expected to depend on homeostatic adaptation to the ensuing lipostatic perturbations and on efficient interorganelle lipid transport. We therefore performed genetic screens to identify factors involved in both of these processes. Among the candidates identified, we find genes linked to transcriptional regulation of lipid homeostasis, lipid metabolism and transport. In particular, we identify a requirement for Csf1 -an uncharacterized protein harboring a Chorein-N lipid transport domain- for survival under certain rewired conditions as well as lipidomic adaptation to cold, implicating Csf1 in interorganelle lipid transport and homeostatic adaptation.

Structural and biochemical insights into lipid transport by VPS13 proteins

“…Such a scenario was recently proposed for a newly identified member of the VPS13 family, Csf1p (Toulmay et al, 2022). We predict that this model of a partnership between scramblases, or other classes of multi-pass integral membrane proteins, and lipid transporters will also be applicable to more recently identified members of the VPS13 protein family, including SHIP164, KIAA1109/Csf1p, and Hobbit/Hob1p/Hob2p (Gomes Castro et al, 2021;Hanna et al, 2021;John Peter et al, 2021;Neuman et al, 2022;Toulmay et al, 2022).…”

Sequence analysis and structural predictions of lipid transfer bridges in the repeating beta groove (RBG) superfamily reveals past and present domain variations affecting form, function and interactions of VPS13, ATG2, SHIP164, Hobbit and Tweek

“…The N-termini of both Hobbit and Tweek start with TMHs that integrate into the ER (Figure 1A) (John Peter et al, 2022;Neuman et al, 2022a;Toulmay et al, 2022). While Hobbit has only a TMH, in Tweek and homologs the TMH is followed by an amphipathic helix (Figure 5E and Supplementary Table 2A).…”

“…Hobbit and Tweek both start with transmembrane helices (TMHs) that anchor them in the ER (Figure 1A) (Castro et al, 2022;Hanna et al, 2022;John Peter et al, 2022;Toulmay et al, 2022). A feature at the N-terminus of Hobbit and Tweek is that their RBG2s are dissimilar to the RBG2 shared by VPS13, ATG2 and SHIP164.…”

Mechanisms of Non-Vesicular Exchange of Lipids at Membrane Contact Sites: Of Shuttles, Tunnels and, Funnels

“…In yeasts deficient for the ER PS synthase, heterologous ectopic expression of bacterial PS synthase targeted to peroxisomes or LDs shows that PS reaches mitochondria to be transformed into PE ( Shiino et al, 2020 ). Another recent study combining the simplification and systematic rewiring of PE/PC biosynthesis in yeast using enzyme retargeting ( Figure 2 ) and saturated transposon mutagenesis analysis has elegantly revealed the robustness of lipid cellular distribution together with the existence of lipostatic adaption mechanisms ( Peter et al, 2021 ) that remain to be studied. Other ERMES functions include involvement in mitochondrial protein import and assembly ( Ellenrieder et al, 2017 ), calcium signaling and regulation of its cellular pools, distribution of mitochondria and their DNA ( Friedman et al, 2011 ; Murley et al, 2013 ), iron homeostasis ( Xue et al, 2017 ) and coenzyme Q biosynthesis ( Eisenberg-Bord et al, 2019 ).…”

“…Studies of the SMP domains of E-SYT2 ( Saheki et al, 2016 ), Mdm12 and Mmm1 in ERMES ( Jeong et al, 2017 ; Kawano et al, 2017 ; AhYoung and Egea, 2019 ), the Ups1/Mdm35 ( Watanabe et al, 2015 ) and also Osh6 ( Maeda et al, 2013 ; Moser von Filseck et al, 2015 ) and OSBP ( Mesmin et al, 2013 ) are examples of these approaches. Other studies have used partially purified intact membrane fractions to monitor lipid transfer ( Kojima et al, 2016 ) or genetic and mutagenesis methods combined to lipidomics to study the effect of lipid biosynthesis rewiring on the levels and distributions of cellular lipids in the network of organelles ( John Peter et al, 2021 ; Peter et al, 2021 ).…”