a, Genetic interaction (E-MAP) profiles were generated for strains harbouring ORM2 deletion, ORM1 over-expression, and ORM2 over-expression alleles (orm2Δ, pTDH3-ORM1 & pTEF2-ORM1, and pTDH3-ORM2 & pTEF2-ORM2, respectively). Correlations between the E-MAP profiles of these strains and those of >1400 other yeast mutants are shown in histograms (see Methods and Supplementary Data). b, Lipids were extracted from the indicated strains and analyzed using a global mass spectrometry approach30. Amounts of the indicated metabolites are shown relative to wild-type (WT; average ± s.d., n = 4; see Methods and Supplementary Data). c, Logarithmic-phase growth rates for the indicated strains were measured in standard media or media supplemented with myriocin at 150 ng/ml. Growth rates (average ± s.d., n ≥ 3) are shown normalized to wild-type (WT); asterisks denote statistical significance (p < 0.05). d, Schematic of the sphingolipid biosynthesis pathway in S. cerevisiae, with selected metabolites and genes indicated. Abbreviations are as follows: FA-CoA (fatty acid co-enzyme A), VLCFA-CoA (very long chain fatty acid co-enzyme A), LCBs (long chain bases), LCB-Ps (long chain base phosphates), IPC (inositolphosphorylceramide), MIPC (mannosyl-inositolphosphorylceramide), and MIP₂C (mannosyl-diinositolphosphorylceramide).

a, HEK293T cells were transfected with either an empty vector or a vector encoding Ormdl3 fused to the 3×Flag epitope (N- or C-terminal fusion). Immunoprecipitations with anti-Flag agarose were analyzed by Western blot against human serine palmitoyltransferase (α-hSptlc1) and against the Flag epitope (α-Flag). b, Hela cells were transfected with siRNAs directed against the indicated genes. After 72 hr, cells were harvested and their lipids were analyzed by mass spectrometry. Ceramide levels normalized to phosphatidylcholine (PC) are shown (relative to untreated cells; average ± s.d., n = 3).

a, Lysates were prepared from strains expressing wild-type (WT) or phospho-site mutant 3×Flag-Orm1 or 3×Flag-Orm2 after growth in standard media or media supplemented with 150 ng/ml myriocin. Western blots are shown after protein separation on phosphate-affinity gels. Residues mutated to alanine in the indicated phospho-mutants are highlighted in red below. b, Native immunoprecipitations of 3×Flag-tagged wild-type or phospho-mutant Orm1 and Orm2 were performed from strains expressing wild-type or phospho-mutant 3×HA-Orm1/2 after growth in standard media or media supplemented with 100 ng/ml myriocin. Western blots were analyzed as in Fig. 4c. c, Model for homeostatic regulation of Orm1/2, in which Orm proteins act as negative regulators of serine palmitoyltransferase (Lcb1/Lcb2/Tsc3), whose inhibitory activity is dependent on levels of downstream sphingolipids. P-Orm1/2 denotes phosphorylated Orm1/2; see Fig. 1d for abbreviations used. d, LCBs were extracted and analyzed from wild-type (WT) and 3×Flag-tagged ORM1/2 double phospho-mutant strains grown in standard media. Data were analyzed as in Fig. 4b (average ± s.d., n = 3). e, Serial dilutions of wild-type (WT), ORM1/2 deletion, and 3×Flag-tagged ORM1/2 double-phospho-mutant strains were spotted on plates with 0 or 280 ng/ml myriocin and imaged after growth for 24–36 hr.

a, Colloidal-stained gels are shown for proteins eluted after affinity purifications from strains expressing 3×Flag-Orm1 or 3×Flag-Orm2, or from an untagged control strain. Immunoprecipitated proteins were identified by mass spectrometry (see Supplementary Table 1; asterisk indicates the partially obscured protein Tsc3, whose presence was confirmed by mass spectrometry). b, Affinity purifications of 3×Flag-Lcb1 from wild-type (WT), sac1Δ, and orm1Δ/orm2Δ strains were performed as in a. Asterisks indicate bands that are lost in deletion backgrounds.

a, Serial dilutions of the indicated strains were spotted on plates with 0, 200, or 400 ng/ml myriocin and imaged after growth for 24–48 hr. b, LCBs were extracted and analyzed from wild-type (WT) and ORM1/2 deletion strains grown in media supplemented with the indicated concentrations of myriocin. The sums of C18 dihydrosphingosine and phytosphingosine peak intensities from n ≥ 2 experiments are shown (relative to wild-type LCB levels in the absence of myriocin). c, Native immunoprecipitations of 3×Flag-tagged Orm1 and Orm2 were performed from strains grown in standard media or media supplemented with 150 ng/ml myriocin and analyzed by Western blot. The indicated strains also expressed 3×HA-Orm1/2 from their endogenous loci (diploid strains were used to examine self-association of Orm1 and Orm2). d, Lysates were prepared from yeast expressing 3×Flag-Orm1/2 after growth in media containing the indicated concentrations of myriocin. Western blots show phosphorylated forms of 3×Flag-Orm1 (P-Orm1) and 3×Flag-Orm2 (P-Orm2) after separation on phosphate-affinity gels35. An immunoprecipitated (IP) sample treated with calf intestine phosphatase (CIP) shows the position of un-phosphorylated Orm1/2.