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Structure of the yeast ceramide synthase

Nature Structural & Molecular Biology volume 32, pages 441–449 (2025)Cite this article

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Abstract

Ceramides are essential lipids involved in forming complex sphingolipids and acting as signaling molecules. They result from the N-acylation of a sphingoid base and a CoA-activated fatty acid, a reaction catalyzed by the ceramide synthase (CerS) family of enzymes. Yet, the precise structural details and catalytic mechanisms of CerSs have remained elusive. Here we used cryo-electron microscopy single-particle analysis to unravel the structure of the yeast CerS complex in both an active and a fumonisin B1-inhibited state. Our results reveal the complex’s architecture as a dimer of Lip1 subunits bound to the catalytic subunits Lag1 and Lac1. Each catalytic subunit forms a hydrophobic crevice connecting the cytosolic site with the intermembrane space. The active site, located centrally in the tunnel, was resolved in a substrate preloaded state, representing one intermediate in ceramide synthesis. Our data provide evidence for competitive binding of fumonisin B1 to the acyl-CoA-binding tunnel.

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Fig. 1: Architecture of the yeast CerS complex.
Fig. 2: Subunit and small-molecule interactions within the CerS.
Fig. 3: Surface properties of the CerS linked to its function.
Fig. 4: Acb1 is important for CerS activity.
Fig. 5: FB1 blocks the cytosolic ligand entry site of the CerS.

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Data availability

All density maps and models were deposited to the EM Data Bank (https://www.ebi.ac.uk/emdb/) and the PDB (https://www.rcsb.org/) and are publicly available under the following accession numbers: PDB 8QTN and EMD-18652 for ScCerS apo; PDB 8QTR and EMD-18653 for FB1-bound ScCerS. The CerS model is available from ModelArchive at https://www.modelarchive.org/doi/10.5452/ma-i2sme. All structural data were visualized with ChimeraX and protein interactions were analyzed with the help of PDBe PISA73. The 2D ligand–protein interaction diagrams were calculated using LigPlotZ74. The MS proteomics data were deposited to the ProteomeXchange Consortium through the PRIDE partner repository and are publicly available (PXD054198 and PXD054237). Other databases used were UniProt (https://www.uniprot.org/), AlphaFold-Multimer (https://cosmic-cryoem.org/tools/alphafoldmultimer/) and AlphaFold. Source data are provided with this paper.

Code availability

No code was generated.

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Acknowledgements

We thank members of the F.F. and A.M. labs for their valuable comments on the manuscript. This work was funded by the German Research Foundation DFG (SFB1557 P6 to F.F., P11 to A.M., FR 3647/2-2, INST 190/201-1 [503478512] and FR 3647/4-1 [491484150] to F.F. and INST.190/196-1 FUGG [455249649], MO2752/3-6 [37802001] and SPRUNG Stay inspired 15-76251-2-04/22 (10743/2022) to A.M.), the BMBF 532 01ED2010 to A.M. and the RISE UP! program of the Boehringer Ingelheim Foundation to F.F. S.V. acknowledges support from the European Research Council under the European Union’s Horizon 2020 research and innovation program (grant agreement no. 803952). J.-H.S. is supported by a fellowship from the Friedrich Ebert foundation.

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Author notes

  1. These authors contributed equally: Jan-Hannes Schäfer, Lena Clausmeyer, Carolin Körner.

Authors and Affiliations

  1. Department of Biology/Chemistry, Structural Biology Section, Osnabrück University, Osnabrück, Germany

    Jan-Hannes Schäfer, Dovile Januliene & Arne Moeller

  2. Department of Biology/Chemistry, Bioanalytical Chemistry Section, Osnabrück University, Osnabrück, Germany

    Lena Clausmeyer, Carolin Körner, Bianca M. Esch, Verena N. Wolf & Florian Fröhlich

  3. Department of Biology, University of Fribourg, Fribourg, Switzerland

    Jennifer Sapia, Yara Ahmed & Stefano Vanni

  4. Center of Cellular Nanoanalytic Osnabrück (CellNanOs), Osnabrück University, Osnabrück, Germany

    Stefan Walter

  5. Swiss National Center for Competence in Research (NCCR), Bio-inspired Materials, University of Fribourg, Fribourg, Switzerland

    Stefano Vanni

  6. Center of Cellular Nanoanalytic Osnabrück (CellNanOs), Osnabrück University, Osnabrück, Germany

    Dovile Januliene, Arne Moeller & Florian Fröhlich

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  1. Jan-Hannes Schäfer
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Contributions

J.H.S., L.C., C.K., V.N.W., B.M.E., S.W., J.S. and Y.A. carried out all experiments. J.H.S., L.C., C.K., V.N.W., B.M.E. and S.W. performed the data analysis. J.H.S., L.C., C.K., J.S., Y.A. and F.F. performed data visualization. F.F., A.M. and D.J. conceptualized the project. J.H.S., L.C., C.K., F.F. and A.M. wrote the original draft of the manuscript. J.H.S., L.C., C.K., S.V., F.F. and A.M. reviewed and edited the text. F.F., A.M. and S.V. secured funding for this study.

Corresponding authors

Correspondence to Arne Moeller or Florian Fröhlich.

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Nature Structural & Molecular Biology thanks Michael Airola and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Primary Handling Editor: Katarzyna Ciazynska, in collaboration with the Nature Structural & Molecular Biology team.

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Extended data

Extended Data Fig. 1 In vitro functional characterization of the yeast ceramide synthase complex.

A Pfam-based ___domain annotation of the CerS subunits. B Summary of the mass spectrometric analysis of the sample GST-Acb1 (E). C Summary of the mass spectrometric analysis of the CerS sample (F). D Size-exclusion chromatography profile of GDN-solubilized CerS complex. E Coomassie-blue stained SDS-PAGE gel of purified GST-Acb1. N = 1. F Coomassie-blue stained SDS-PAGE gel of GDN-solubilized CerS complex of indicated fraction from (D). N = 1. G Representative micrograph and 2D class averages from negative-stain TEM. 100 nm scale bar. 36 micrographs were taken and 4170 partikel are used for the 2D classes. H Relative CerS enzyme activity measurement with or without the specific CerS-inhibitor fumonisin B1 (FB1). Control with addition of BSA and the Fatty-acid-Coenzyme A transport protein Acb1. n = 3 technically independent samples for H and data are presented as mean values ± SD. Exact P-values are shown in Supplementary Table 4. I Label free proteomics of yeast cells expressing Lag1-msGFP2 compared to untagged control cells. In the volcano plot, the protein abundance ratios of Lag1-msGFP2 over control cells are plotted against the negative log10 of the P-value of the two-tailed t-test for each protein. J Chemical structures of FB1 (Fumonisin-B1), PHS (phytosphingosine) and C26:0 (hexacosanoic acid).

Source data

Extended Data Fig. 2 Cryo-EM analysis and validation of the ceramide synthase apo complex.

A Processing workflow for the CerS in its apo state. All processing steps were performed in cryoSPARC. Representative cryo-EM micrograph and 2D-class averages are given. 50 nm scale bar in micrograph. B FSC-curve with gold-standard criterion at 0.134 and 3.0 Å overall resolution. C Local resolution estimation (threshold 0.16). D Angular particle distribution.

Extended Data Fig. 3 Cryo-EM analysis and validation of the FB1-bound ceramide synthase complex.

A Processing workflow for the FB1-bound CerS complex. All processing steps were performed in cryoSPARC. Representative cryo-EM micrograph and 2D-class averages are given. 50 nm scale bar in micrograph. B FSC-curve with gold-standard criterion at 0.134 and 3.2 Å overall resolution. C Local resolution estimation (threshold 0.16). D Angular particle distribution.

Extended Data Fig. 4 Local cryo-EM density map quality of apo and FB1-bound ceramide synthase.

Side-by-side comparison of selected residues and ligands within all cryo-EM density maps of A Lag1 B FB1 (Fumonisin-B1) C Lac1, D Lip1, E Lip1*, F C26:0-His255 (Lag1/Lac1) G PI16:0-lipid (phosphoinositide) and ammonium ion NH4 + , H PE18:0-lipid (phosphoethanolamine). Contouring levels set to 0.16.

Extended Data Fig. 5 Ligand interaction diagram of ceramide synthase.

2D ligand interaction diagram of: A C26:0 in the apo-state with Lag1, B C26:0 in the apo-state with Lac1, C C26:0 in the FB1-bound state with Lag1, D C26:0 in the FB1-bound state with Lac1, E FB1-binding to Lag1, F FB1-binding to Lac1, G PI16:0 binding to Lac1 in the apo-state and H PI16:0 binding to Lac1 in the FB1-bound state. I PI16:0 binding to Lip1 in FB1-bound state and Lip1* in J. Diagrams were calculated and visualized with LigPlot+1.

Extended Data Fig. 6 Analysis of Lac1 Mutants.

Purification of a Lac1-FLAG-Lip1 homodimer analyzed by SDS-PAGE for A Lac1-WT B Lac1_5F C Lac1_PP D Lac1 K389A R392A. Each purification N = 1. E-H Proteomic analysis of the purified complex. Unique peptides of each protein identified are shown in the tables. I Specific CerS enzyme activity measurement in the presence of the fatty-acid-Coenzyme A transport protein Acb1 for a heterodimeric Lac1-Lip1 complex and the respective Lac1_5F and Lac1_PP mutants. n = 3 technically independent samples for I and data are presented as mean values ± SD. Exact P-values are shown in Supplementary Table 4. J Analysis of the co-purified long chain bases with the Lac1_WT-Lip1 heterodimeric complex or the Lac1_5F-Lip1 heterodimeric complex. n = 4 technically independent samples for J and data are presented as mean values ± SD. Exact P-values are shown in Supplementary Table 5. K Tetrad analysis of lac1Δ (blue) lag1Δ (green) cells, expressing Lac1_5F (upper panel) in dark grey and Lac1_PPWLac1_PP (lower panel) in light grey.

Source data

Extended Data Fig. 7 Analysis of Lip1 Mutants.

Purification of the heterodimeric CerS complex analyzed by SDS-PAGE for A Lip1_F40R and B Lip1_W50A_F51A. Each purifications N = 1. C Proteomic analysis of the purified complex with Lip1_F40R and in D with Lip1_W50A_F51A. Unique peptides of each protein identified are shown in the tables. E Relative CerS enzyme activity measurement in the presence of the fatty-acid-Coenzyme A transport protein Acb1 for the CerS with either WT Lip1, Lip1_F40R or Lip1_W50A_F51A. n = 3 technically independent samples for E and data are presented as mean values ± SD. Exact P-values are shown in Supplementary Table 4. F Levels of phytosphingosine (PHS), in WT cells, lip1Δ cells expressing LIP1_F40R or lip1Δ cells. n = 3 technically independent samples for F and data are presented as mean values ± SD. Exact P-values are shown in Supplementary Table 8. G Tetrad analysis of lip1Δ (orange) cells, expressing LIP1_WT (upper panel, brown), or LIP1_F40R (lower panel, sand).

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Extended Data Fig. 8 Structural conservation of the yeast ceramide synthase.

A Cartoon representation of Lac1 color-coded by sequence-conservation with data from Supplementary Fig. 10a. B Cartoon representation of Lag1 color-coded by sequence-conservation with data from Supplementary Fig. 11. Yellow areas denote regions of no conservation. The catalytic residues are highlighted as spheres. C Luminal view of the cartoon-representation of superimposed Lag1 (yellow) and Lag1 (grey), color-coded by Cα root-mean-square deviation (r.s.m.d.). Catalytic residues and C26:0 are shown as sticks. D Pair-based sequence alignment of Lac1 Saccharomyces cerevisiae (P28496) and Lag1 Saccharomyces cerevisiae (P38703). Sequences are color coded by conservation with a cut-off at 30 %. Catalytic residues are highlighted in red. Non-resolvable residues from cryo-EM are marked in light gray. Alignment was prepared with Jalview. E Comparison of local densities in Lag1 and Lac1 for density-based subunit differentiation. The corresponding sequences are highlighted in yellow in the alignment (D).

Extended Data Fig. 9 AlphaFold2 multimer prediction of the yeast ceramide synthase.

A AlphaFold2 multimer prediction of the yeast CerS (ScLag1: P38703, ScLac1: P28496, ScLip1: Q03579) shown as cartoon-representation, color-coded by the predicted local distance difference test (pLDDT). The low-confidence termini of each subunit are annotated accordingly. Distance measurement between Cα of Lag1-D390 and Lac1-D394 from their respective proposed dimerization motif (DXRSDXE). B Validation parameters of the prediction, including the pLDDT and the PAE (prediction aligned error matrix). The pLDDT plot is color-coded according to the different subunits. The prediction was run locally using AlphaFold v.2.1.0 (ref. 67) in the multimer-mode (template date set to 2020-05-14).

Extended Data Fig. 10 Sequence conservation of the yeast ceramide synthase subunits Lac1 and Lip1.

A Clustal-based multiple-sequence alignment (MSA) of Lac1 Saccharomyces cerevisiae (P28496), Lag1 Arabidopsis thaliana (Q9LDF2), CS hyl-1 Caenorhabditis elegans (G5ED45), TLC ___domain containing protein Caenorhabditis elegans (X5LPS5), CS Dictyostelium discoideum (Q54S87), CS4 Mus musculus (Q9D6J1), CS2-like Danio rerio (E7F9X7), CS2 Homo sapiens (Q96G23), CS1 Homo sapiens (P27544). Sequences are color coded by conservation with a cut-off at 30 %. Catalytic residues are highlighted in red. Sequences were selected by highest sequence identity hits from protein BLAST of selected model organisms. B Sequence of Saccharomyces cerevisiae Lip1 (Q03579). Non-resolvable residues from cryo-EM are marked in light gray. MSAs were prepared with Jalview3.

Supplementary information

Supplementary Information

Supplementary Figs. 1 and 2 and Tables 1–9.

Reporting Summary

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Source Data Fig. 4

Statistical source data.

Source Data Fig. 4

Unprocessed plates of tetrad dissections.

Source Data Extended Data Fig. 1

Statistical source data.

Source Data Extended Data Fig. 1

Unprocessed Coomassie gels.

Source Data Extended Data Fig. 6

Statistical source data.

Source Data Extended Data Fig. 6

Unprocessed Coomassie gels and unprocessed plates of tetrad dissections.

Source Data Extended Data Fig. 7

Unprocessed Coomassie gels and unprocessed plates of tetrad dissections.

Source Data Extended Data Fig. 7

Statistical source data.

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Schäfer, JH., Clausmeyer, L., Körner, C. et al. Structure of the yeast ceramide synthase. Nat Struct Mol Biol 32, 441–449 (2025). https://doi.org/10.1038/s41594-024-01415-2

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