Despite the essential roles of sphingolipids both as structural components of membranes and critical signalling molecules, we have a limited understanding of how cells sense and regulate their levels. Here we reveal the function in sphingolipid metabolism of the ORM genes (known as ORMDL genes in humans)—a conserved gene family that includes ORMDL3, which has recently been identified as a potential risk factor for childhood asthma. Starting from an unbiased functional genomic approach in Saccharomyces cerevisiae, we identify Orm proteins as negative regulators of sphingolipid synthesis that form a conserved complex with serine palmitoyltransferase, the first and rate-limiting enzyme in sphingolipid production. We also define a regulatory pathway in which phosphorylation of Orm proteins relieves their inhibitory activity when sphingolipid production is disrupted. Changes in ORM gene expression or mutations to their phosphorylation sites cause dysregulation of sphingolipid metabolism. Our work identifies the Orm proteins as critical mediators of sphingolipid homeostasis and raises the possibility that sphingolipid misregulation contributes to the development of childhood asthma.
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We acknowledge A. Falick, S. Zhou and D. King for identification of proteins by mass spectrometry; M. Schuldiner and S. Wang for E-MAP data collection; D. Fiedler and K. Shokat for providing the phosphate-binding acrylamide reagent; E. Griffis and M. D’Ambrosio for assistance with fluorescence microscopy; N. Ingolia for a codon-optimized mCherry-tagging plasmid; E. Burchard and J. Galanter for discussions of asthma genetics; I. Poser and A. Hyman for advice and providing the HeLa-Kyoto cell line; and M. Bassik, G. Brar, V. Denic, A. Frost, N. Ingolia, E. Quan, B. Toyama and other members of the Weissman laboratory for discussions. This work was supported by funding from Deutsche Forschungsgemeinschaft SFB/TR 13 projects A1 (K.S.) and D1 (A.S.), EUFP6 PRISM (K.S.), ETH Zurich (R.A.), the National Heart, Lung, and Blood Institute, National Institute of Health (N01-HV-28179) (R.A.), SystemsX.ch, the Swiss initiative for systems biology (R.A.), the Boehringer Ingelheim Fonds and the Swiss National Science Foundation (B.B.), the Howard Hughes Medical Institute (J.S.W.), the University of California, San Francisco Strategic Asthma Basic Research Center (J.S.W.), the National Science Foundation Graduate Research Fellowship Program (D.K.B.) and the Fannie and John Hertz Foundation (D.K.B.).
Author Contributions D.K.B. designed, performed and interpreted experiments. S.R.C. oversaw E-MAP data collection and analysis. B.B. performed and analysed protein mass spectrometry experiments to identify sites of phosphorylation under the supervision of R.A. C.S.E. performed and analysed lipidomic measurements with the support of A.S. and K.S. J.S.W. designed and interpreted experiments. D.K.B. and J.S.W. prepared the manuscript.
The authors declare no competing financial interests.
This file contains Supplementary Figures 1-7 with Legends, Supplementary Methods, Supplementary Table 1 and Supplementary References. (PDF 16508 kb)
This file contains genetic interaction (E-MAP) data including the genetic interaction scores obtained for select mutant strains with all other strains in our E-MAP dataset. Correlation values obtained for pair-wise comparisons of interaction profiles are also included. (XLS 425 kb)
This file contains additional information on lipid species quantified by mass spectrometry for lipidomic experiments performed on yeast and Hela cell samples. (XLS 158 kb)
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Breslow, D., Collins, S., Bodenmiller, B. et al. Orm family proteins mediate sphingolipid homeostasis. Nature 463, 1048–1053 (2010). https://doi.org/10.1038/nature08787
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