The membrane transporter SLC25A48 enables transport of choline into human mitochondria

Abstract

Choline has important physiological functions as a precursor for essential cell components, signaling molecules, phospholipids, and the neurotransmitter acetylcholine. Choline is a water-soluble charged molecule requiring transport proteins to cross biological membranes. Although transporters continue to be identified, membrane transport of choline is incompletely understood and knowledge about choline transport into intracellular organelles such as mitochondria remains limited. Here we show that SLC25A48 imports choline into human mitochondria. Human loss-of-function mutations in SLC25A48 show impaired choline transport into mitochondria and are associated with elevated urine and plasma choline levels. Thus, our studies may have implications for understanding and treating conditions related to choline metabolism. Choline has important physiological functions as a precursor for essential cell components, signaling molecules, phospholipids, and the neurotransmitter acetylcholine. Choline is a water-soluble charged molecule requiring transport proteins to cross biological membranes. Although transporters continue to be identified, membrane transport of choline is incompletely understood and knowledge about choline transport into intracellular organelles such as mitochondria remains limited. Here we show that SLC25A48 imports choline into human mitochondria. Human loss-of-function mutations in SLC25A48 show impaired choline transport into mitochondria and are associated with elevated urine and plasma choline levels. Thus, our studies may have implications for understanding and treating conditions related to choline metabolism. Translational StatementThis study uncovers the function of a human protein called solute carrier SLC25A48 by showing that it acts as a membrane transporter for choline. Choline has important physiological functions as a building block for essential cell components and signaling molecules. Choline is a water-soluble charged molecule and, therefore, requires transport proteins to cross biological membranes. How it gets into cellular compartments, like mitochondria, has been poorly understood. This study shows that SLC25A48 transports choline into mitochondria. Mutations in SLC25A48 disrupt choline transport into mitochondria, leading to higher levels of choline in urine and blood in humans. These findings shed light on how choline is handled within cells and could have implications for understanding and treating conditions related to choline metabolism. This study uncovers the function of a human protein called solute carrier SLC25A48 by showing that it acts as a membrane transporter for choline. Choline has important physiological functions as a building block for essential cell components and signaling molecules. Choline is a water-soluble charged molecule and, therefore, requires transport proteins to cross biological membranes. How it gets into cellular compartments, like mitochondria, has been poorly understood. This study shows that SLC25A48 transports choline into mitochondria. Mutations in SLC25A48 disrupt choline transport into mitochondria, leading to higher levels of choline in urine and blood in humans. These findings shed light on how choline is handled within cells and could have implications for understanding and treating conditions related to choline metabolism. Membrane transport proteins play a crucial role in the movement of ions and metabolites across biological membranes. Their proper functioning is essential for many physiological processes. Despite significant progress in our understanding of these proteins, a substantial number of them remain uncharacterized, often referred to as orphan membrane transport proteins.1Meixner E. Goldmann U. Sedlyarov V. et al.A substrate-based ontology for human solute carriers.Mol Syst Biol. 2020; 16: e9652Crossref PubMed Scopus (31) Google Scholar Deorphanization efforts are essential to unveil the hidden cellular functions and roles of these proteins in health and disease. Recent large-scale genome-wide association studies of metabolite levels provide links between common genetic variants in membrane transporter-encoding genes and metabolite levels.2Schlosser P. Scherer N. Grundner-Culemann F. et al.Genetic studies of paired metabolomes reveal enzymatic and transport processes at the interface of plasma and urine.Nat Genet. 2023; 55: 995-1008Crossref PubMed Scopus (11) Google Scholar,3Suhre K. Shin S.Y. Petersen A.K. et al.Human metabolic individuality in biomedical and pharmaceutical research.Nature. 2011; 477: 54-60Crossref PubMed Scopus (837) Google Scholar This generates testable hypotheses to identify the physiological substrates of established and orphan human transport proteins in vivo. Choline is an essential nutrient with important roles in a variety of physiological processes and metabolic pathways. It is an essential component of phospholipids in cell membranes, such as phosphatidylcholines, a precursor of the neurotransmitter acetylcholine and of the osmoregulatory betaine, and an important player in lipid metabolism.4Ueland P.M. Choline and betaine in health and disease.J Inherit Metab Dis. 2011; 34: 3-15Crossref PubMed Scopus (448) Google Scholar Recent studies have uncovered a growing body of evidence linking choline to various diseases, such as neurologic disorders, metabolic syndromes, and liver disease.4Ueland P.M. Choline and betaine in health and disease.J Inherit Metab Dis. 2011; 34: 3-15Crossref PubMed Scopus (448) Google Scholar Several choline transport proteins have been identified and characterized in model systems, including SLC5A7, SLC44A1, SLC44A2, SLC44A4, SLC49A1, and SLC49A2.5Apparsundaram S. Ferguson S.M. George Jr., A.L. et al.Molecular cloning of a human, hemicholinium-3-sensitive choline transporter.Biochem Biophys Res Commun. 2000; 276: 862-867Crossref PubMed Scopus (157) Google Scholar, 6Bennett J.A. Mastrangelo M.A. Ture S.K. et al.The choline transporter Slc44a2 controls platelet activation and thrombosis by regulating mitochondrial function.Nat Commun. 2020; 11: 3479Crossref PubMed Scopus (42) Google Scholar, 7Kenny T.C. Khan A. Son Y. et al.Integrative genetic analysis identifies FLVCR1 as a plasma-membrane choline transporter in mammals.Cell Metab. 2023; 35: 1057-1071.e12Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar, 8Michel V. Bakovic M. The solute carrier 44A1 is a mitochondrial protein and mediates choline transport.FASEB J. 2009; 23: 2749-2758Crossref PubMed Scopus (56) Google Scholar, 9Traiffort E. O'Regan S. Ruat M. The choline transporter-like family SLC44: properties and roles in human diseases.Mol Aspects Med. 2013; 34: 646-654Crossref PubMed Scopus (70) Google Scholar, 10Cater R.J. Mukherjee D. Gil-Iturbe E. et al.Structural and molecular basis of choline uptake into the brain by FLVCR2.Nature. 2024; 629: 704-709Crossref PubMed Scopus (2) Google Scholar However, it is unclear which transporters affect systemic levels of free choline in humans and if they are linked to human genetic variation. pDONR221_SLC25A48 was a kind gift from the RESOLUTE Consortium (Addgene plasmid number 131995; http://n2t.net/addgene:131995; research resource identifier [RRID]: Addgene_131995). The SLC25A48 transcript was ligated into the pcDNA6.flag vector, and mutations were generated by site-directed mutagenesis (see Supplementary Methods for details). HEK293T and HeLa cell lines were cultured as described previously.11Wegierski T. Steffl D. Kopp C. et al.TRPP2 channels regulate apoptosis through the Ca2+ concentration in the endoplasmic reticulum.EMBO J. 2009; 28: 490-499Crossref PubMed Scopus (94) Google Scholar HEK293T cells were transfected with the calcium phosphate method, and HeLa cells with FuGENE HD (Promega E2311). Proteins were isolated, separated using sodium dodecylsulfate–polyacrylamide gel electrophoresis, and transferred to polyvinylidene difluoride membranes, as described previously.12Hofherr A. Seger C. Fitzpatrick F. et al.The mitochondrial transporter SLC25A25 links ciliary TRPP2 signaling and cellular metabolism.PLoS Biol. 2018; 16e2005651Crossref PubMed Scopus (17) Google Scholar Antibodies were anti-Flag (Sigma Aldrich F3165) and ß-actin (Sigma Aldrich A1978). Chemiluminescence signals were captured with the Intas ChemoCam system. Western blot analyses are representative of 3 experiments with similar results (Supplementary Methods).Figure 2Rare damaging variants in SLC25A48 impair choline transport. (a) Comparison of inverse normal transformed choline levels in urine and plasma among carriers (N = 47) and noncarriers (N = 4572 for urine; N = 4666 for plasma) of putative rare damaging driver variants in SLC25A48 (P value by unpaired t-test [2 tailed]: 3.8 × 10-21 for urine and 1.3 × 10-07 for plasma). (b) Localization of rare, damaging driver variants with respect to their protein position in SLC25A48 (Q6ZT89, corresponding to transcript ENST00000681962.1, domains based on InterPro, x axis). Symbol shape corresponds to variant consequence, and the size represents the positive effect size of each individual variant on urine choline levels (Supplementary Table S1). Individual variant association –log10(P value) with urine choline levels is shown on the y axis. The 4 variants with the largest effects on urine choline levels were selected for subsequent functional analyses and are labeled. (c) Western blot analysis of wild-type versus mutant flag-tagged SLC25A48. Actin was used as the loading control. (d) Cellular localization of the SLC25A48 missense mutation p.R179P compared with wild-type SLC25A48. Indirect immunofluorescence of SLC25A48-flag and cytochrome C oxidase subunit 4 (COX4) as mitochondrial marker. Colors in merged image: SLC25A48 (turquoise), COX4 (violet), and 4′,6-diamidino-2-phenylindole (blue). Bar = 5 μM. (e) Quantification of colocalization of SLC25A48 and COX4 (Pearson correlation coefficient) shows a significant reduction of mitochondrial localization of SLC25A48-R179P compared with wild type (∗∗∗∗P < 0.0001; see Supplementary Figure S7 for other mutants). (f) Relative mitochondrial uptake of radiolabeled 3H-choline in cells expressing wild-type and mutant SLC25A48. Transport of mutant SLC25A48 was normalized to wild-type transport. ∗P < 0.05, ∗∗∗∗P < 0.0001. (g) Position of damaging mutations in model of SLC25A48 (AlphaFold) in intermembrane space (IMS)–facing conformation (helices numbered). Left: side chains of mutant residues are highlighted in pink. The protein part that would be truncated in p.R243∗ is in pink. Center, right: residues of the conserved matrix-salt-bridge network (dotted line), including D27 (mutation in p.D27G) are highlighted in red and blue sticks for acidic and basic residues, respectively. Mito_carr, mitochondrial carrier protein repeat; SOLCAR, solute carrier repeat. To optimize viewing of this image, please see the online version of this article at www.kidney-international.org.View Large Image Figure ViewerDownload Hi-res image Download (PPT) HeLa cells were prepared for indirect immunofluorescence, as described in the Supplementary Methods. Confocal imaging was conducted on a Zeiss LSM980 MP AiryScan 2 microscope with a 63× objective. Quantitative colocalization analysis was performed with the Colocalization plugin in the Zeiss ZEN blue 3.4 software (Supplementary Methods). HEK293T cells were grown and transfected in 500-cm2 plates. Two days after transfection, cells were harvested and mitochondrial pellets were obtained.13Wieckowski M.R. Giorgi C. Lebiedzinska M. et al.Isolation of mitochondria-associated membranes and mitochondria from animal tissues and cells.Nat Protoc. 2009; 4: 1582-1590Crossref PubMed Scopus (681) Google Scholar Mitochondrial pellets were resuspended in a mitochondrial uptake buffer (KCl, 120 mM; sucrose, 25 mM; N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid, 10 mM; ethyleneglycol-bis-[β-aminoethylether]-N,N,N′,N′-tetraacetic acid, 1 mM; KH2PO4, 1 mM; MgCl2, 5 mM; glutamate, 15 mM; and malate, 7.5 mM, adjusted to a pH 7.2)6Bennett J.A. Mastrangelo M.A. Ture S.K. et al.The choline transporter Slc44a2 controls platelet activation and thrombosis by regulating mitochondrial function.Nat Commun. 2020; 11: 3479Crossref PubMed Scopus (42) Google Scholar (see Supplementary Methods). A total of 25 μg of mitochondria was suspended in 50 μl mitochondrial uptake buffer with subsequent addition of 2× choline buffer (mitochondrial uptake buffer supplemented with 20 μM choline chloride and 10 nM choline chloride [methyl-3H]) and incubated for 5 minutes. Samples were washed twice with ice-cold mitochondria washing buffer (mitochondrial uptake buffer with 20 μM choline chloride), and the pellet was resuspended in Ultima Gold scintillation cocktail for quantification of 3H-choline content using liquid scintillation counting. GraphPad Prism 9.5.1 software was used to graph, analyze, and present the obtained data. All results are expressed as mean ± SEM. All experiments were independently performed at least 3 times. A 2-tailed Mann-Whitney test was used to calculate P values when the sample size (biological replicates) was at least 10, and an unpaired t-test was used when the sample size was <10. P < 0.05 was considered significant. Genetic associations with plasma and urine choline levels, measured as part of the Metabolon HD4 Global Discovery metabolomics panel, were evaluated in the German Chronic Kidney Disease study.14Eckardt K.U. Barthlein B. Baid-Agrawal S. et al.The German Chronic Kidney Disease (GCKD) study: design and methods.Nephrol Dial Transplant. 2012; 27: 1454-1460Crossref PubMed Scopus (127) Google Scholar The SLC25A48 gene was investigated using whole-exome sequencing data. Details about the study sample, choline measurements, and whole-exome sequencing (sequencing, alignment, and variant calling) are found in the Supplementary Methods. Called variants were annotated using Variant Effect Predictor version 109, incorporating various tools to predict deleteriousness.15Ioannidis N.M. Rothstein J.H. Pejaver V. et al.REVEL: an ensemble method for predicting the pathogenicity of rare missense variants.Am J Hum Genet. 2016; 99: 877-885Abstract Full Text Full Text PDF PubMed Scopus (1368) Google Scholar, 16Rentzsch P. Witten D. Cooper G.M. et al.CADD: predicting the deleteriousness of variants throughout the human genome.Nucleic Acids Res. 2019; 47: D886-D894Crossref PubMed Scopus (2026) Google Scholar, 17Liu X. Li C. Mou C. et al.dbNSFP v4: a comprehensive database of transcript-specific functional predictions and annotations for human nonsynonymous and splice-site SNVs.Genome Med. 2020; 12: 103Crossref PubMed Scopus (269) Google Scholar Loss-of-function variants for SLC25A48 were evaluated using the LoFtee Variant Effect Predictor plugin.18Karczewski K.J. Francioli L.C. Tiao G. et al.The mutational constraint spectrum quantified from variation in 141,456 humans.Nature. 2020; 581: 434-443Crossref PubMed Scopus (5266) Google Scholar Two masks ("LoF_mis" and "HI_mis") that included rare variants (minor allele frequency <1%) were generated for variant aggregation testing. The LoF_mis mask included high-confidence loss-of-function variants, missense variants with a MetaSVM score >0, or in-frame nonsynonymous variants with a fathmm-XF-coding score >0.5, whereas the HI_mis mask encompassed variants with high-impact consequences or missense variants meeting specific criteria. Burden tests, adjusting for covariates, were performed using the seqMeta R-package version 1.6.7. Statistical significance was defined as P < 0.05. Single-variant, covariate-adjusted association tests were conducted under additive modeling. Results are presented for the HI_mis mask, which yielded lower association P values. Further details are presented in the Supplementary Methods. Methods related to the association of rare SLC25A48 variants with gene expression, as well as human traits and diseases from the UK Biobank (application identifier 64806), are found in the Supplementary Methods. The SLC25A48 AlphaFold19Jumper J. Evans R. Pritzel A. et al.Highly accurate protein structure prediction with AlphaFold.Nature. 2021; 596: 583-589Crossref PubMed Scopus (16733) Google Scholar model was retrieved from the AlphaFold Protein Structure Database hosted by the European Molecular Biology Laboratories - European Bioinformatics Institute (EMBL-EBI; https://alphafold.ebi.ac.uk, accession code Q6ZT89). To identify potential homologous proteins with experimental structures available, a sequence-based BLAST search of the Protein Data Bank as well as a structural homology–based search using the DALI Server20Holm L. Laiho A. Toronen P. et al.DALI shines a light on remote homologs: one hundred discoveries.Protein Sci. 2023; 32e4519Crossref PubMed Scopus (127) Google Scholar were performed. Details about the structures of other SLC25 family members, how they were superimposed, and how the matrix-open structure was modeled can be found in the Supplementary Methods. Figures were prepared using the Molecular found through genome-wide association studies of metabolite levels that common SLC25A48 variants are associated with choline levels in urine P. Scherer N. Grundner-Culemann F. et al.Genetic studies of paired metabolomes reveal enzymatic and transport processes at the interface of plasma and urine.Nat Genet. 2023; 55: 995-1008Crossref PubMed Scopus (11) Google Scholar and established that the genetic basis of this association is with plasma choline as well as SLC25A48 transcript levels in brain (Supplementary Figure the that the orphan solute carrier SLC25A48 may be a choline SLC25A48 transcript levels are in brain and the is in cells of the of the a cell type in mitochondria with functions such as of (Supplementary Figure SLC25A48 to mitochondria in human cells Supplementary Figures and therefore, mitochondria of cells SLC25A48 and measured mitochondrial uptake of radiolabeled choline from cells SLC25A48 a significant of choline uptake compared with mitochondria from cells of SLC25A48 mitochondrial choline uptake and choline concentration (Supplementary Figure Mitochondrial uptake of choline was and concentration and These the that SLC25A48 is a identified, mitochondrial choline SLC25A48 transports choline at and at physiological of free choline in humans E. et of choline in and plasma using a PubMed Scopus Google Scholar To test is a between choline levels in humans and impaired mitochondrial choline we investigated the of rare, damaging variants in SLC25A48 and found significant associations with urine and plasma choline levels test P = × and × Supplementary Table of driver variants (Supplementary higher levels of choline compared with which was in urine in plasma driver variants into of SLC25A48 The association with choline was metabolite other choline significant genetic associations after for (Supplementary Figure Choline has been in various human from neurologic diseases to metabolic is evidence linking genetic in choline transport to human disease. therefore, putative loss-of-function mutations in SLC25A48 were associated with of the human traits and diseases in the UK Biobank that are related to tissues SLC25A48 is expressed of the (Supplementary Table or (Supplementary Table traits was associated with SLC25A48 putative loss-of-function carrier after for testing. To variants were related to choline levels, we generated 4 mutations in the SLC25A48 using site-directed mutagenesis and these variants in human cell lines to study their effect on protein expression, and Western blot analyses protein of investigated SLC25A48 mutations compared with wild-type protein mutations from mitochondria and with a significant reduction of the mitochondrial colocalization compared with wild-type SLC25A48 and Supplementary Figure whereas other mutations to mitochondria like wild-type transporters and Supplementary Figure These show that of the investigated SLC25A48 mutations in protein measured choline uptake in mitochondria to which mutations affect SLC25A48 transport Despite in and SLC25A48 mutations impaired mitochondrial choline uptake These that impaired mitochondrial choline transport SLC25A48 results in plasma and urine choline levels in humans. This is by the concentration of choline in cells (Supplementary Figure which the for choline into cells plasma membrane choline a our that the investigated mutations loss-of-function through including expression, and impaired with normal and To a understanding of specific mutations impaired we structural of SLC25A48 in 2 Supplementary Figure The SLC25A48 were in with experimental structures of other SLC25 family members, such as the carrier and mitochondrial protein (Supplementary Figure D27 is part of a matrix-salt-bridge network of conserved residues, which is a component of the of these transporters and, essential for transport This is in with the impaired choline transport of normal P. et al.Structural basis of of human protein 2023; Scopus Google The SLC25 mitochondrial carrier structure and Sci. 2020; Full Text Full Text PDF PubMed Scopus Google Scholar The other mutations were on the side of the transporter and may affect and A of this study is the of experiments with SLC25A48. studies SLC25A48 in may provide into the the and the function of this mitochondrial we show that the physiological function of SLC25A48 in humans is choline into mitochondria. Loss-of-function mutations in SLC25A48 impair choline transport into mitochondria and higher levels of free choline in urine and The of SLC25A48 molecular function in humans and studies role in health and disease. All the All generated or in this study were included in the and the Supplementary for this that the findings of this study and gel of Western blot analyses are from the corresponding on The for SLC25A48 was by the RESOLUTE The and for and would like to the for with and cell and for with The of and was by German identifier was by was by for the of and was by The of was by and urine metabolomics in the German Chronic Kidney Disease (GCKD) study were by metabolomics has from the 2 under The from the European research and and the European of and and the of results the the is for that may be of the it sequencing was by and of was by their for and The study was and is by the and and the for are for the of the to in the The of the study of the various is the number of provide for the and with the Download with Supplementary Download with Supplementary

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