Abstract
Phosphatidylcholine and phosphatidylethanolamine, the two most abundant phospholipids in mammalian cells, are synthesized de novo by the Kennedy pathway from choline and ethanolamine, respectively1,2,3,4,5,6. Despite the essential roles of these lipids, the mechanisms that enable the cellular uptake of choline and ethanolamine remain unknown. Here we show that the protein encoded by FLVCR1, whose mutation leads to the neurodegenerative syndrome posterior column ataxia and retinitis pigmentosa7,8,9, transports extracellular choline and ethanolamine into cells for phosphorylation by downstream kinases to initiate the Kennedy pathway. Structures of FLVCR1 in the presence of choline and ethanolamine reveal that both metabolites bind to a common binding site comprising aromatic and polar residues. Despite binding to a common site, FLVCR1 interacts in different ways with the larger quaternary amine of choline in and with the primary amine of ethanolamine. Structure-guided mutagenesis identified residues that are crucial for the transport of ethanolamine, but dispensable for choline transport, enabling functional separation of the entry points into the two branches of the Kennedy pathway. Altogether, these studies reveal how FLVCR1 is a high-affinity metabolite transporter that serves as the common origin for phospholipid biosynthesis by two branches of the Kennedy pathway.
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Data availability
Cryo-EM maps have been deposited in the Electron Microscopy Data Bank (EMDB) under the accession codes EMD-42107 (choline-bound FLVCR1), EMD-42108 (ethanolamine-bound FLVCR1), EMD-42109 (endogenous choline-bound FLVCR1), EMD-42110 (endogenous choline-bound FLVCR1, from images collected in 1 mM ethanolamine) and EMD-42111 (endogenous ligand-bound FLVCR1). Atomic coordinates have been deposited in the Protein Data Bank (PDB) under the accession codes 8UBW (choline-bound FLVCR1), 8UBX (ethanolamine-bound FLVCR1), 8UBY (endogenous choline-bound FLVCR1), 8UBZ (endogenous choline-bound FLVCR1, from images collected in 1 mM ethanolamine) and 8UC0 (endogenous ligand-bound FLVCR1). The atomic coordinates of previously published structures of E. coli SotB (PDB 6KKL) in an inward-facing state and E. coli DgoT (PDB 6E9N) in an inward-facing state were used in this study. Co-dependencies between FLVCR1 and all genes computed from CRISPR DepMap Chronos 2023Q2 used in this study were downloaded from https://depmap.org/portal/all. Source data are provided with this paper.
Code availability
The code written to perform the FLVCR1 DepMap coessentiality analysis is available at https://github.com/artemkhan/Coessentiality_DepMAP_FLVCR1.git.
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Acknowledgements
We thank M. J. de la Cruz and the Simons Electron Microscopy Center staff for help with data acquisition; the Memorial Sloan Kettering Cancer Center High Performance Computing group for assistance with data processing; the members of the laboratories for comments on the manuscript; and L. Finley for discussions. R.K.H. is supported by the National Institutes of Health (NIH) National Cancer Institute Cancer Center Support Grant P30-CA008748 and is a Searle Scholar. T.C.K. is supported by the NIH National Institute of Diabetes and Digestive and Kidney Diseases (F32 DK127836), the Shapiro-Silverberg Fund for the Advancement of Translational Research and a Merck Postdoctoral Fellowship at The Rockefeller University. K.B. is supported by the NIH National Institute of Diabetes and Digestive and Kidney Diseases (R01 DK123323-01), and a Mark Foundation Emerging Leader Award, and is a Searle Scholar and a Pew-Stewart Scholar. Some of this work was carried out at the Simons Electron Microscopy Center at the New York Structural Biology Center, with major support from the Simons Foundation (SF349247).
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Conceptualization: Y.S. and R.K.H.; methodology: Y.S., T.C.K., A.K., K.B. and R.K.H.; formal analysis: Y.S. and R.K.H.; investigation, Y.S., T.C.K., K.B. and R.K.H.; writing (original draft): Y.S. and R.K.H.; funding acquisition, T.C.K., K.B. and R.K.H.
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R.K.H. is a consultant for F. Hoffmann-La Roche. K.B. is a scientific adviser to Nanocare Pharmaceuticals and Atavistik Bio. The other authors declare no competing interests.
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Extended data figures and tables
Extended Data Fig. 1 Regulation of FLVCR1-mediated choline uptake.
a, Background subtracted uptake of 20 nM [methyl-3H]choline by vesicles containing FLVCR1 after 30 min in buffer at pH 4.5-10.5. Background was measured by uptake of 20 nM [methyl-3H]choline into the protein-free control liposomes. n = 3 technically independent samples. b, Background subtracted uptake of 20 nM [methyl-3H]choline by FLVCR1 proteoliposome after 30 min in buffer in the presence or absence of Na+, K+, Ca2+, or Mg2+. Background was measured by uptake of 20 nM [methyl-3H]choline into the protein-free control liposomes. n = 3 technically independent samples.
Extended Data Fig. 2 Validation of cryo-EM structures of FLVCR1.
a-c Representative cryo-EM images and two-dimensional class averages of human FLVCR1 in 1 mM choline (a), 1 mM ethanolamine (b) or without added substrate (c). Number of collected cryo-EM images are shown. d-h, Plots showing Fourier shell correlations between two independent half-maps (black) and between density-modified map and refined atomic model (red) for choline-bound FLVCR1 (d), ethanolamine-bound FLVCR1 (e), endogenous choline-bound FLVCR1 obtained from FLVCR1 incubated with 1 mM ethanolamine (f), endogenous choline-bound FLVCR1 obtained from FLVCR1 without exogenous ligand incubation (g), and endogenous ligand-bound FLVCR1 obtained from FLVCR1 without exogenous ligand incubation (h). Dashed lines are indicated at FSC = 0.5 and FSC = 0.143. i-m, Local resolution plots of choline-bound FLVCR1 (i), ethanolamine-bound FLVCR1 (j), endogenous-choline bound FLVCR1 obtained from FLVCR1 incubated with 1 mM ethanolamine (k), endogenous choline-bound FLVCR1 obtained from FLVCR1 without exogenous substrate incubation (l), and endogenous ligand-bound FLVCR1 obtained from FLVCR1 without exogenous ligand incubation (m).
Extended Data Fig. 3 Cryo-EM analysis of hFLVCR1.
Flow-chart summarizing Cryo-EM image acquisition and processing of human FLVCR1 without added substrate (a), in 1 mM choline (b), or 1 mM ethanolamine (c). Number of collected cryo-EM images and selected particles are shown.
Extended Data Fig. 4 Choline-bound FLVCR1 adopts an inward-facing conformation.
a, Superposition of TM1-6 and TM7-12. RMSD = 3.5 Å. b, Central cavity viewed from the cytosolic side. Aspartate and glutamate residues in and near the entrance to the central cavity are shown as sticks. c, Central section of choline-bound FLVCR1 is contiguous with choline shown as sticks. d, Blue spheres depict the minimum radius of the central cavity in the choline-bound state as a function of position. e-f, Superposition of choline-bound FLVCR1 (blue) with E. coli SotB (D; PDB: 6KKL; RMSD = 2.3 Å; red) (e) or E. coli DgoT (E; PDB: 6E9N; RMSD = 2.6 Å; cyan) (f).
Extended Data Fig. 5 Sequence alignment of SLC49A family members and identified disease-associated substitutions.
a, Sequence alignment of human FLVCR1 (SLC49A1), human FLVCR2 (SLC49A2), human MFSD7 (SLC49A3), human DIRC2 (SLC49A4) and Drosophila CG1358 performed using Clustal Omega54 and pyBoxshade (https://github.com/mdbaron42/pyBoxshade). Substrate-binding site residues are highlighted by red boxes. Residues whose substitution leads to PCARP or Fowler syndrome are highlighted by blue and green boxes, respectively. b, Structure of choline-bound FLVCR1 (left) and Alphafold2 model of FLVCR2 (right)55 with residues whose mutation leads to PCARP and Fowler syndrome highlighted in blue and green, respectively. FLVCR1 substrate binding site residues and the corresponding residues in FLVCR2 are highlighted in red.
Extended Data Fig. 6 Cryo-EM structures of FLVCR1 determined in a substrate-free condition.
a-b, Cryo-EM density maps and atomic models of FLVCR1 in endogenous choline (a) and endogenous ligand-bound (b) states. c-d, Central slice of the central cavity of choline-bound (c) and endogenous ligand-bound (d) states, with surface colored white. Choline is shown as sticks in c. Endogenous ligand is not modeled or shown in d. e, Superposition of choline-bound (grey) and endogenous choline-bound (blue) states. f, Superposition of substrate-binding sites in choline-bound (grey) and endogenous choline-bound (blue) states.
Extended Data Fig. 7 Cryo-EM structures of FLVCR1 determined in 1mM ethanolamine.
a-b, Cryo-EM density maps of endogenous choline-bound FLVCR1 (a) and ethanolamine-bound FLVCR1 (b) states, determined from particles imaged in the presence of 1 mM ethanolamine. c, Superposition of ethanolamine-bound (gold) and choline-bound (blue) states. d, Substrate-binding site in endogenous choline-bound FLVCR1 state, determined from particles imaged in the presence of 1 mM ethanolamine. Residues and modelled substrates are shown as sticks. Density is shown as a grey isosurface and contoured at 3.0 σ. e-f, Coordination of ethanolamine in the substrate-binding site in ethanolamine-bound state (e) and choline in the substrate-binding site in the choline-bound state (f). Polar interactions are shown as dashed lines and distances as solid lines.
Extended Data Fig. 8 FLVCR1 substrate-binding site mutants.
a, FSEC analysis of wild-type or substrate-binding site mutants fused to mCerulean expressed in FLVCR1-knockout HEK293T cells. b, Western blot analysis of FLVCR1-knockout HEK293T cells expressing a vector control or wild-type or mutant FLVCR1 cDNA. GAPDH was ran on a separate gel as sample processing controls. For western blot source data, see Supplementary Fig. 1. c, Cumulative log2 fold change in cell number of HEK293T cells and FLVCR1-knockout HEK293T cells expressing a vector control or wild-type or mutant FLVCR1 cDNA. n = 3 biologically independent samples. (P-values: HEK293T WT and HEK293T FLVCR1 KO + FLVCR1 WT cDNA; p = 3x10−3, HEK293T FLVCR1 KO + FLVCR1 WT cDNA and HEK293T FLVCR1 KO + FLVCR1 Q214A cDNA; p = 9x10−1, HEK293T FLVCR1 KO + Vector and HEK293T FLVCR1 KO + FLVCR1 W125A cDNA; p = 9x10−1, HEK293T FLVCR1 KO + Vector and HEK293T FLVCR1 KO + FLVCR1 Y153A cDNA; p = 1x10−1, HEK293T FLVCR1 KO + FLVCR1 WT cDNA and HEK293T FLVCR1 KO + Vector; p = 8x10−5).
Extended Data Fig. 9 Incorporation of labeled choline into betaine requires FLVCR1.
Schematic for tracing [1,2-13C2] choline into betaine (left) and the abundance of betaine M + 2 after incubation with 21.5 µM [1,2-13C2] choline for 1 h in FLVCR1-knockout HEK293T cells expressing a vector control or wild-type or mutant FLVCR1 cDNA (right). n = 3 biologically independent samples.
Supplementary information
Supplementary Fig. 1
Raw, uncropped western blot image of Extended Data Fig. 8b. GAPDH was run on a separate gel as a sample processing control.
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Son, Y., Kenny, T.C., Khan, A. et al. Structural basis of lipid head group entry to the Kennedy pathway by FLVCR1. Nature 629, 710–716 (2024). https://doi.org/10.1038/s41586-024-07374-4
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DOI: https://doi.org/10.1038/s41586-024-07374-4
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