Structure of the ceramide-bound SPOTS complex

Sphingolipids are structural membrane components that also function in cellular stress responses. The serine palmitoyltransferase (SPT) catalyzes the rate-limiting step in sphingolipid biogenesis. Its activity is tightly regulated through multiple binding partners, including Tsc3, Orm proteins, ceramides, and the phosphatidylinositol-4-phosphate (PI4P) phosphatase Sac1. The structural organization and regulatory mechanisms of this complex are not yet understood. Here, we report the high-resolution cryo-EM structures of the yeast SPT in complex with Tsc3 and Orm1 (SPOT) as dimers and monomers and a monomeric complex further carrying Sac1 (SPOTS). In all complexes, the tight interaction of the downstream metabolite ceramide and Orm1 reveals the ceramide-dependent inhibition. Additionally, observation of ceramide and ergosterol binding suggests a co-regulation of sphingolipid biogenesis and sterol metabolism within the SPOTS complex.

f Specific SPT enzyme activity measurement with or without the specific SPT-inhibitor myriocin (Myr).g Ergosterol quantification from GDN-solubilized and purified SPOTS complex.h Ceramide quantification from GDN-solubilized and purified SPOTS complex and Orm-free SPT-Tsc3-Sac1 complex.n=4 technically independent samples for f-h and data are presented as mean values ± SD.Source data are provided as a Source Data file for b, eh.

Sup-Fig 2: Cryo-EM analysis of the SPT-Orm1 complexes
Processing workflow for SPOT-dimer, SPOT-monomer and SPOTS complex.All processing steps were performed in cryoSPARC.Representative cryo-EM micrograph and 2D-class averages.100 nm scale bar in micrograph and 20 nm scale bar in 2D class-averages.Contouring levels according to Supplementary Figure 3.

Sup-Fig 1 :
In vitro functional characterization of the yeast SPOTS complex a Pfam-based domain annotation of the SPOTS subunits.b SEC profile and corresponding Coomassie-blue stained SDS-PAGE gel of indicated fraction of GDN-solubilized SPOTS complex.c Representative micrograph and 2D class averages from negative-stain TEM. 100 nm scale bar.d Summary of the massspectrometric analysis of the sample in b. e ALFA-Lcb1 (left) or FLAG-Lcb1 (right) were pulled down from diploid cells expressing one ALFA-tagged and one FLAG-tagged version of Lcb1.Western blots were probed with antibodies against the ALFA and the FLAG tag.I = input; FT = flow-through; W3 = wash, E = eluate.

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Cryo-EM map evaluation Local resolution estimation and angular particle distribution of a SPOT-dimer (level 0.13), b masked SPOTdimer (level 0.13), c SPOTS (level 0.18) and d SPOT-monomer (level 0.14).Local cryo-EM density map quality of SPOT and SPOTS complexes Side-by-side comparison of selected residues and ligands within all cryo-EM density maps of a Orm1, b Lcb1, c Tsc3, d Lcb2, Sac1 and ligands in e. Ergosterol (ERG) was not present in maps denoted with an x.

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Ligand interaction diagram of SPOT and SPOTS complexes 2D ligand interaction diagram of ceramide 44:0;4 (a), internal aldimine between PLP and Lcb2 K366 (b), ergosterols (ERG) (c) and glyco-diosgenin (GDN) (d).Key interacting residues are shown as ball and sticks with polar contacts given as green dotted lines.Diagrams were calculated and visualized with LigPlot+ 1 .(a,b,d for all oligomers, c for SPOT-monomer and SPOTS).a Identification of four amphipathic helices in SPOTS involves Lcb1 I18-Q35 , Lcb2 Y58-F85 , Tsc3 G26-H45 and Sac1 D476-T490 .b Physico-chemical properties, including the mean hydrophobic moment < µH > (arrow proportional, perpendicular to membrane plane) and hydrophobicity H were calculated with HeliQuest 2 .Helix wheels are colored according to their chemical polarity.Structural conservation within different SPOT oligomers a Side-by-side comparison of the Cα distances between the conserved proline residue in TM1 in the yeast SPOT-dimer and human SPT-ORMDL3 dimer (PDB: 7K0M 3 ).Adjacent subunits are colored in blue and yellow for clarity.b Superposition of SPT-ORMDL3 and SPOT-dimer results in a global root-mean-square deviation (RMSD) of 4.8 Å. c Overview and superposition of ORMDL3-free human SPT (PDB: 7K0I 3 ) and the SPOT-dimer results in a global RMSD of 2.8 Å. d Superposition of Arabidopsis thaliana (PDB: 7YJM 4 , gray) and SPOT-monomer with a local Cα RMSD between the Orm1 subunits of 1.0 Å. Yeast Orm1 in orange and Lcb2 in green.Orm1 N-termini depicted as spheres.e Cα RMSD between the Sac1 subunit from SPOTS and an AlphaFold prediction of Dictyostelium discoideum (Q55AW9).f Sequence conservation between yeast Sac1 and its homolog from D. discoideum.The Lcb2 subunit (green) is added for highlighting the interface.Superposition of two SPOT-monomers (blue and yellow) onto the SPOT-dimer (grey).The sterical clash of Lcb1-TM0a is indicated with an arrow.h Superposition of an AlphaFold prediction of D. discoideum (Q55AW9, gray) and the structure of the SPOTS complex (violet).Sup-Fig 8: Micelle curvature induction by SPT-ORMDL3 and yeast SPT oligomers Representative volume slices of a SPOTS b SPOT-monomer c SPOT-dimer d SPT-ORMDL3 (PDB: 7K0M, EMD-22602 3 ) and e SPTLC1/SPLTC2/ssSPTa (PDB: 7K0I, EMD-22598 3 ).EMDB maps were gaussian filtered by 1.5-fold standard deviations.9: Sequence conservation and unique features within different SPT complexes Clustal-based multiple-sequence alignment (MSA) of a Sac1 from S. cerevisiae (P32368), H. sapiens (Q9NTJ5) and D. discoideum (Q55AW9) with the C-terminal β-hairpin region and Lcb2 interacting loop T98-D105 highlighted in green.b Comparison of S. cerevisiae Orm1 (P53224) and Orm2 (Q06144).The elongated N-terminal region of Orm1 is colored in red and the mutated phosphorylation-sites S51-53 are marked in green.c Comparison of H. sapiens ORMDL3 (Q8N138), A. thaliana Orm1 (Q9C5I0) and yeast Orm1 (P53224).The N-terminal methionine is highlighted in green, which was observed to the substrate binding tunnel in human SPT-ORMDL3 (PDB: 7K0M).AtOrm1 Y5-A8 adopts a β-sheet with AtLcb2a I2-P5 (PDB: 7YJM); both highlighted in green.The Orm1 helix F63-L71 interacts with Lcb1/2 is highlighted in green.d Yeast Lcb1 (P25045) has an elongated N-terminal region, including helix TM0a (T20-Q35) and TM0b (Q40-S49), which are not present in human SPTLC1 (O15269).e Alignment of Lcb2 homologues from human SPTLC2 (O15270) and Arabidopsis Lcb2a (Q9LSZ9) with yeast Lcb2 (P40970).The PATP-loop and catalytic lysine are highlighted in green.Non-resolvable residues from cryo-EM are marked in light gray.Sequences are color coded by conservation with a cut-off at 30 %. MSAs were prepared with Jalview 5 .Sup-Fig.10: The N-terminus of Lcb1 faces the cytosol A cytosolic GFP-tagged ALFA nanobody (ALFA-NB-GFP) is expressed in control cells (upper panels), ALFA-Lcb1 cells (middle panels) and ALFA-Lcb2 cells (lower panels) also expressing DsRed-HDEL.Both, ALFA-Lcb1 and ALFA-Lcb2 allow the recruitment of the otherwise cytosolic ALFA-NB-GFP to the ER membrane marked with DsRed-HDEL showing that the N-termini of both proteins face the cytosol.GFP (left panels), DsRed-HDEL (middle left panels), merged images (middle right panels) and brightfield images (right panels) are shown.Scale bar = 5 µm.Source data are provided as a Source Data file.) Sup-Fig.11: Estimation of compositional heterogeneity Scale-value color-coded cryo-EM densities for the overall reconstruction and local density for 44:0;4 ceramide are shown.a SPOT-dimer.b SPOT-dimer masked.c SPOT-monomer and d SPOTS-complex.All maps are contoured automatically by OccuPy 6 .Low compositional heterogeneity is shown in blue, high values are shown in red.Sup-Fig.12: The TM0 helix affects SPT activity in vivo but not complex formation a Purification of SPOTS complex harboring the lcb1T21-Q35 (left) or lcb1T21-S49 mutants.SDS-PAGE gels after elution with FLAG peptide are shown.b Total LCB and ceramide levels were measured in cells expressing either the WT ALFA-tagged Lcb1 or the ALFA-lcb1T21-Q35 mutant.Data were analyzed using