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Calcium-dependent phospholipid scrambling by TMEM16F

Abstract

In all animal cells, phospholipids are asymmetrically distributed between the outer and inner leaflets of the plasma membrane1. This asymmetrical phospholipid distribution is disrupted in various biological systems. For example, when blood platelets are activated, they expose phosphatidylserine (PtdSer) to trigger the clotting system2,3. The PtdSer exposure is believed to be mediated by Ca2+-dependent phospholipid scramblases that transport phospholipids bidirectionally1,4, but its molecular mechanism is still unknown. Here we show that TMEM16F (transmembrane protein 16F) is an essential component for the Ca2+-dependent exposure of PtdSer on the cell surface. When a mouse B-cell line, Ba/F3, was treated with a Ca2+ ionophore under low-Ca2+ conditions, it reversibly exposed PtdSer. Using this property, we established a Ba/F3 subline that strongly exposed PtdSer by repetitive fluorescence-activated cell sorting. A complementary DNA library was constructed from the subline, and a cDNA that caused Ba/F3 to expose PtdSer spontaneously was identified by expression cloning. The cDNA encoded a constitutively active mutant of TMEM16F, a protein with eight transmembrane segments5. Wild-type TMEM16F was localized on the plasma membrane and conferred Ca2+-dependent scrambling of phospholipids. A patient with Scott syndrome6,7, which results from a defect in phospholipid scrambling activity8,9, was found to carry a mutation at a splice-acceptor site of the gene encoding TMEM16F, causing the premature termination of the protein.

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Figure 1: Molecular cloning of TMEM16F.
Figure 2: Phospholipid scrambling in TMEM16F-expressing cells.
Figure 3: Requirement of TMEM16F for phospholipid scrambling.
Figure 4: A splice mutation of TMEM16F in a patient with Scott syndrome.

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Acknowledgements

We thank T. Wiedmer for critical reading of our manuscript, and M. Fujii and M. Harayama for secretarial assistance. This work was supported in part by Grants-in-Aid for Specially Promoted Research (to S.N.) and for Young Scientists Start-up (to J.S.) from the Japan Society for the Promotion of Science. J.S. is supported by a research fellowship from the Japan Society for the Promotion of Science. P.J.S. was supported by grants from the National Institutes of Health, USA.

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J.S. designed and performed the experiments, and wrote the manuscript. M.U. provided biotin-labelled Ro09-0198 peptide. P.J.S. provided EBV-transformed cell lines from a patient and the patient’s parents, and commented on the manuscript. S.N. was responsible for the overall study design and for writing the manuscript.

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Correspondence to Shigekazu Nagata.

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The authors declare no competing financial interests.

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Suzuki, J., Umeda, M., Sims, P. et al. Calcium-dependent phospholipid scrambling by TMEM16F. Nature 468, 834–838 (2010). https://doi.org/10.1038/nature09583

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