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Natl. Acad. Sci. U.S.A."],"published-print":{"date-parts":[[2023,3,7]]},"abstract":"<jats:p>Major Facilitator Superfamily Domain containing 2a (Mfsd2a) is a sodium-dependent lysophosphatidylcholine (LPC) transporter expressed at the blood\u2013brain barrier that constitutes the main pathway by which the brain obtains omega-3 fatty acids, such as docosahexanoic acid. Mfsd2a deficiency in humans results in severe microcephaly, underscoring the importance of LPC transport by Mfsd2a for brain development. Biochemical studies and recent cryo-electron microscopy (cryo-EM) structures of Mfsd2a bound to LPC suggest that Mfsd2a transports LPC via an alternating access mechanism between outward-facing and inward-facing conformational states in which the LPC inverts during transport between the outer and inner leaflet of a membrane. However, direct biochemical evidence of flippase activity by Mfsd2a has not been demonstrated and it is not understood how Mfsd2a could invert LPC between the outer and inner leaflet of the membrane in a sodium-dependent manner. Here, we established a unique in\u00a0vitro assay using recombinant Mfsd2a reconstituted in liposomes that exploits the ability of Mfsd2a to transport lysophosphatidylserine (LPS) coupled with a small molecule LPS binding fluorophore that allowed for monitoring of directional flipping of the LPS headgroup from the outer to the inner liposome membrane. Using this assay, we demonstrate that Mfsd2a flips LPS from the outer to the inner leaflet of a membrane bilayer in a sodium-dependent manner. Furthermore, using cryo-EM structures as guides together with mutagenesis and a cell-based transport assay, we identify amino acid residues important for Mfsd2a activity that likely constitute substrate interaction domains. These studies provide direct biochemical evidence that Mfsd2a functions as a lysolipid flippase.<\/jats:p>","DOI":"10.1073\/pnas.2215290120","type":"journal-article","created":{"date-parts":[[2023,2,27]],"date-time":"2023-02-27T15:17:27Z","timestamp":1677511047000},"update-policy":"https:\/\/doi.org\/10.1073\/pnas.cm10313","source":"Crossref","is-referenced-by-count":43,"title":["Mfsd2a utilizes a flippase mechanism to mediate omega-3 fatty acid lysolipid transport"],"prefix":"10.1073","volume":"120","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-9763-0652","authenticated-orcid":false,"given":"Geok-Lin","family":"Chua","sequence":"first","affiliation":[{"name":"Signature Research Program in Cardiovascular and Metabolic Disorders, Duke-National University of Singapore Medical School, Singapore 169857, Singapore"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-6399-8814","authenticated-orcid":false,"given":"Bryan C.","family":"Tan","sequence":"additional","affiliation":[{"name":"Signature Research Program in Cardiovascular and Metabolic Disorders, Duke-National University of Singapore Medical School, Singapore 169857, Singapore"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-0819-3479","authenticated-orcid":false,"given":"Randy Y. J.","family":"Loke","sequence":"additional","affiliation":[{"name":"Signature Research Program in Cardiovascular and Metabolic Disorders, Duke-National University of Singapore Medical School, Singapore 169857, Singapore"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-5375-9454","authenticated-orcid":false,"given":"Menglan","family":"He","sequence":"additional","affiliation":[{"name":"Signature Research Program in Cardiovascular and Metabolic Disorders, Duke-National University of Singapore Medical School, Singapore 169857, Singapore"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-6753-9228","authenticated-orcid":false,"given":"Cheen-Fei","family":"Chin","sequence":"additional","affiliation":[{"name":"Signature Research Program in Cardiovascular and Metabolic Disorders, Duke-National University of Singapore Medical School, Singapore 169857, Singapore"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-8981-1376","authenticated-orcid":false,"given":"Bernice H.","family":"Wong","sequence":"additional","affiliation":[{"name":"Signature Research Program in Cardiovascular and Metabolic Disorders, Duke-National University of Singapore Medical School, Singapore 169857, Singapore"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-4122-0259","authenticated-orcid":false,"given":"Alvin C. Y.","family":"Kuk","sequence":"additional","affiliation":[{"name":"Signature Research Program in Cardiovascular and Metabolic Disorders, Duke-National University of Singapore Medical School, Singapore 169857, Singapore"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-3920-568X","authenticated-orcid":false,"given":"Mei","family":"Ding","sequence":"additional","affiliation":[{"name":"Singapore Lipidomics Incubator, Life Sciences Institute, National University of Singapore, Singapore 117456, Singapore"},{"name":"Department of Biochemistry, Yong Loo Lin School of Medicine, National University of Singapore, Singapore 117596, Singapore"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Markus R.","family":"Wenk","sequence":"additional","affiliation":[{"name":"Singapore Lipidomics Incubator, Life Sciences Institute, National University of Singapore, Singapore 117456, Singapore"},{"name":"Department of Biochemistry, Yong Loo Lin School of Medicine, National University of Singapore, Singapore 117596, Singapore"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-2274-361X","authenticated-orcid":false,"given":"Lan","family":"Guan","sequence":"additional","affiliation":[{"name":"Department of Cell Physiology and Molecular Biophysics, Center for Membrane Protein Research, School of Medicine, Texas Tech University Health Sciences Center, Lubbock, TX 79430"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-7545-7484","authenticated-orcid":false,"given":"Federico","family":"Torta","sequence":"additional","affiliation":[{"name":"Singapore Lipidomics Incubator, Life Sciences Institute, National University of Singapore, Singapore 117456, Singapore"},{"name":"Department of Biochemistry, Yong Loo Lin School of Medicine, National University of Singapore, Singapore 117596, Singapore"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-7289-9890","authenticated-orcid":false,"given":"David L.","family":"Silver","sequence":"additional","affiliation":[{"name":"Signature Research Program in Cardiovascular and Metabolic Disorders, Duke-National University of Singapore Medical School, Singapore 169857, Singapore"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"341","published-online":{"date-parts":[[2023,2,27]]},"reference":[{"key":"e_1_3_4_1_2","doi-asserted-by":"publisher","DOI":"10.1038\/nature13241"},{"key":"e_1_3_4_2_2","doi-asserted-by":"publisher","DOI":"10.1074\/jbc.M116.721340"},{"key":"e_1_3_4_3_2","doi-asserted-by":"publisher","DOI":"10.1074\/jbc.M116.721035"},{"key":"e_1_3_4_4_2","doi-asserted-by":"publisher","DOI":"10.4049\/jimmunol.1801585"},{"key":"e_1_3_4_5_2","doi-asserted-by":"publisher","DOI":"10.1074\/jbc.M116.721340"},{"key":"e_1_3_4_6_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.jbc.2022.101709"},{"key":"e_1_3_4_7_2","doi-asserted-by":"publisher","DOI":"10.1371\/journal.pone.0050629"},{"key":"e_1_3_4_8_2","doi-asserted-by":"publisher","DOI":"10.1126\/science.abl4896"},{"key":"e_1_3_4_9_2","doi-asserted-by":"publisher","DOI":"10.1038\/ng.3313"},{"key":"e_1_3_4_10_2","doi-asserted-by":"publisher","DOI":"10.1038\/ng.3311"},{"key":"e_1_3_4_11_2","doi-asserted-by":"crossref","unstructured":"T. Harel Homozygous mutation in MFSD2A encoding a lysolipid transporter for docosahexanoic acid is associated with microcephaly and hypomyelination. Neurogenetics 19 227\u2013235 (2018) 10.1007\/s10048-018-0556-6.","DOI":"10.1007\/s10048-018-0556-6"},{"key":"e_1_3_4_12_2","doi-asserted-by":"crossref","unstructured":"M. Scala Biallelic MFSD2A variants associated with congenital microcephaly developmental delay and recognizable neuroimaging features. Eur. J. Hum. 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