Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

Cryo-EM structure of the human L-type amino acid transporter 1 in complex with glycoprotein CD98hc

Nature Structural & Molecular Biology volume 26, pages 510–517 (2019)Cite this article

Subjects

Abstract

The L-type amino acid transporter 1 (LAT1 or SLC7A5) transports large neutral amino acids across the membrane and is crucial for brain drug delivery and tumor growth. LAT1 forms a disulfide-linked heterodimer with CD98 heavy chain (CD98hc, 4F2hc or SLC3A2), but the mechanism of assembly and amino acid transport are poorly understood. Here we report the cryo-EM structure of the human LAT1–CD98hc heterodimer at 3.3-Å resolution. LAT1 features a canonical Leu T-fold and exhibits an unusual loop structure on transmembrane helix 6, creating an extended cavity that might accommodate bulky amino acids and drugs. CD98hc engages with LAT1 through the extracellular, transmembrane and putative cholesterol-mediated interactions. We also show that two anti-CD98 antibodies recognize distinct, multiple epitopes on CD98hc but not its glycans, explaining their robust reactivities. These results reveal the principles of glycoprotein-solute carrier assembly and provide templates for improving preclinical drugs and antibodies targeting LAT1 or CD98hc.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Fig. 1: Architecture of LAT1–CD98hc.
Fig. 2: Structure of LAT1.
Fig. 3: Structural basis for broad substrate specificity.
Fig. 4: Anti-CD98 antibodies and epitopes.
Fig. 5: Model of amino acid antiport by LAT1–CD98hc.

Similar content being viewed by others

Data availability

The atomic coordinates of LAT1–CD98hc–MEM-108 Fab and CD98hc-ED–HBJ127 Fab–MEM-108 Fab have been deposited in the Protein Data Bank under accession numbers 6JMQ and 6JMR. The cryo-EM density maps have been deposited in the Electron Microscopy Data Bank under accession numbers EMD-9849 and EMD-9850. The raw micrographs used for this study are available at the Electron Microscopy Public Image Archive under accession codes EMPIAR-10264 and EMPIAR-10265. Source data for Fig. 3d,e and Supplementary Figs. 1d–g and 5a–c are available online. All other data are available from the corresponding author upon request.

References

  1. Kandasamy, P., Gyimesi, G., Kanai, Y. & Hediger, M. A. Amino acid transporters revisited: new views in health and disease. Trends Biochem. Sci. 43, 752–789 (2018).

    Article  CAS  Google Scholar 

  2. Kanai, Y. et al. Expression cloning and characterization of a transporter for large neutral amino acids activated by the heavy chain of 4F2 antigen (CD98). J. Biol. Chem. 273, 23629–23632 (1998).

    Article  CAS  Google Scholar 

  3. Mastroberardino, L. et al. Amino-acid transport by heterodimers of 4F2hc/CD98 and members of a permease family. Nature 395, 288–291 (1998).

    Article  CAS  Google Scholar 

  4. Fotiadis, D., Kanai, Y. & Palacín, M. The SLC3 and SLC7 families of amino acid transporters. Mol. Aspects Med. 34, 139–158 (2013).

    Article  CAS  Google Scholar 

  5. Jack, D. L., Paulsen, I. T. & Saier, M. H. The amino acid/polyamine/organocation (APC) superfamily of transporters specific for amino acids, polyamines and organocations. Microbiology 146, 1797–1814 (2000).

    Article  CAS  Google Scholar 

  6. Palacín, M. & Kanai, Y. The ancillary proteins of HATs: SLC3 family of amino acid transporters. Pflügers Arch. 447, 490–494 (2004).

    Article  Google Scholar 

  7. Palacin, M., Errasti-Murugarren, E. & Rosell, A. Heteromeric amino acid transporters. In search of the molecular bases of transport cycle mechanisms. Biochem. Soc. Trans. 44, 745–752 (2016).

    Article  CAS  Google Scholar 

  8. Yanagida, O. et al. Human L-type amino acid transporter 1 (LAT1): characterization of function and expression in tumor cell lines. Biochim. Biophys. Acta Biomembr. 1514, 291–302 (2001).

    Article  CAS  Google Scholar 

  9. Ritchie, J. W. A. & Taylor, P. M. Role of the System L permease LAT1 in amino acid and iodothyronine transport in placenta. Biochem. J. 356, 719–725 (2001).

    Article  CAS  Google Scholar 

  10. Kageyama, T. et al. The 4F2hc/LAT1 complex transports l-DOPA across the blood–brain barrier. Brain Res. 879, 115–121 (2000).

    Article  CAS  Google Scholar 

  11. Uchino, H. et al. Transport of amino acid-related compounds mediated by L-type amino acid transporter 1 (LAT1): insights into the mechanisms of substrate recognition. Mol. Pharmacol. 61, 729–737 (2002).

    Article  CAS  Google Scholar 

  12. Nii, T. et al. Molecular events involved in up-regulating human Na+-independent neutral amino acid transporter LAT1 during T-cell activation. Biochem. J. 358, 693–704 (2001).

    Article  CAS  Google Scholar 

  13. Boado, R. J., Li, J., Nagaya, M., Zhang, C. & Pardridge, W. M. Selective expression of the large neutral amino acid transporter at the blood–brain barrier. Proc. Natl Acad. Sci. USA 96, 12079–12084 (1999).

    Article  CAS  Google Scholar 

  14. Jin, S.-E., Jin, H.-E. & Hong, S.-S. Targeting L-type amino acid transporter 1 for anticancer therapy: clinical impact from diagnostics to therapeutics. Expert Opin. Ther. Targets 19, 1319–1337 (2015).

    Article  CAS  Google Scholar 

  15. Häfliger, P. et al. The LAT1 inhibitor JPH203 reduces growth of thyroid carcinoma in a fully immunocompetent mouse model. J. Exp. Clin. Cancer Res. 37, 234 (2018).

    Article  Google Scholar 

  16. Wiriyasermkul, P. et al. Transport of 3-fluoro-l-α-methyl-tyrosine by tumor-upregulated L-type amino acid transporter 1: a cause of the tumor uptake in PET. J. Nucl. Med. 53, 1253–1261 (2012).

    Article  CAS  Google Scholar 

  17. Oda, K. et al. L-type amino acid transporter 1 inhibitors inhibit tumor cell growth. Cancer Sci. 101, 173–179 (2010).

    Article  CAS  Google Scholar 

  18. Rosilio, C. et al. L-type amino-acid transporter 1 (LAT1): a therapeutic target supporting growth and survival of T-cell lymphoblastic lymphoma/T-cell acute lymphoblastic leukemia. Leukemia 29, 1253–1266 (2015).

    Article  CAS  Google Scholar 

  19. Puris, E., Gynther, M., Huttunen, J., Petsalo, A. & Huttunen, K. M. L-type amino acid transporter 1 utilizing prodrugs: how to achieve effective brain delivery and low systemic exposure of drugs. J. Control. Release 261, 93–104 (2017).

    Article  CAS  Google Scholar 

  20. Nicklin, P. et al. Bidirectional transport of amino acids regulates mTOR and autophagy. Cell 136, 521–534 (2009).

    Article  CAS  Google Scholar 

  21. Fenczik, C. A., Sethi, T., Ramos, J. W., Hughes, P. E. & Ginsberg, M. H. Complementation of dominant suppression implicates CD98 in integrin activation. Nature 390, 36349 (1997).

    Article  Google Scholar 

  22. Cantor, J. et al. CD98hc facilitates B cell proliferation and adaptive humoral immunity. Nat. Immunol. 10, 412–419 (2009).

    Article  CAS  Google Scholar 

  23. Jungnickel, K. E., Parker, J. L. & Newstead, S. Structural basis for amino acid transport by the CAT family of SLC7 transporters. Nat. Commun. 9, 550 (2018).

    Article  Google Scholar 

  24. Fang, Y. et al. Structure of a prokaryotic virtual proton pump at 3.2 Å resolution. Nature 460, 1040–1043 (2009).

    Article  CAS  Google Scholar 

  25. Gao, X. et al. Mechanism of substrate recognition and transport by an amino acid antiporter. Nature 463, 828–832 (2010).

    Article  CAS  Google Scholar 

  26. Kowalczyk, L. et al. Molecular basis of substrate-induced permeation by an amino acid antiporter. Proc. Natl Acad. Sci. USA 108, 3935–3940 (2011).

    Article  CAS  Google Scholar 

  27. Ilgü, H. et al. Insights into the molecular basis for substrate binding and specificity of the wild-type L-arginine/agmatine antiporter AdiC. Proc. Natl Acad. Sci. USA 113, 10358–10363 (2016).

    Article  Google Scholar 

  28. Fort, J. et al. The structure of human 4F2hc ectodomain provides a model for homodimerization and electrostatic interaction with plasma membrane. J. Biol. Chem. 282, 31444–31452 (2007).

    Article  CAS  Google Scholar 

  29. Calonge, M. et al. Cystinuria caused by mutations in rBAT, a gene involved in the transport of cystine. Nat. Genet. 6, 420–425 (1994).

    Article  CAS  Google Scholar 

  30. Feliubadaló, L. et al. Non-type I cystinuria caused by mutations in SLC7A9, encoding a subunit (bo, +AT) of rBAT. Nat. Genet. 23, 52–57 (1999).

    Article  Google Scholar 

  31. Chairoungdua, A. et al. Identification of an amino acid transporter associated with the cystinuria-related type II membrane glycoprotein. J. Biol. Chem. 274, 28845–28848 (1999).

    Article  CAS  Google Scholar 

  32. Borsani, G. et al. SLC7A7, encoding a putative permease-related protein, is mutated in patients with lysinuric protein intolerance. Nat. Genet. 21, 297–301 (1999).

    Article  CAS  Google Scholar 

  33. Tărlungeanu, D. C. et al. Impaired amino acid transport at the blood brain barrier is a cause of autism spectrum disorder. Cell 167, 1481 (2016).

    Article  Google Scholar 

  34. Sigworth, F. J. Principles of cryo-EM single-particle image processing. Microscopy 65, 57–67 (2016).

    Article  Google Scholar 

  35. Wu, S. et al. Fabs enable single particle cryoEM studies of small proteins. Structure 20, 582–592 (2012).

    Article  CAS  Google Scholar 

  36. Yagita, H., Masuko, T. & Hashimoto, Y. Inhibition of tumor cell growth in vitro by murine monoclonal antibodies that recognize a proliferation-associated cell surface antigen system in rats and humans. Cancer Res. 46, 1478–1484 (1986).

    CAS  PubMed  Google Scholar 

  37. Rosell, A. et al. Structural bases for the interaction and stabilization of the human amino acid transporter LAT2 with its ancillary protein 4F2hc. Proc. Natl Acad. Sci. USA 111, 2966–2971 (2014).

    Article  CAS  Google Scholar 

  38. Jeckelmann, J.-M. & Fotiadis, D. Volta phase plate cryo-EM structure of the human heterodimeric amino acid transporter 4F2hc-LAT2. Int. J. Mol. Sci. 20, 931 (2019).

    Article  Google Scholar 

  39. Verrey, F. et al. CATs and HATs: the SLC7 family of amino acid transporters. Pflügers Arch. 447, 532–542 (2004).

    Article  CAS  Google Scholar 

  40. Schweikhard, E. S. & Ziegler, C. M. Current topics in membranes. Curr. Top. Membr. 70, 1–28 (2012).

    Article  CAS  Google Scholar 

  41. Krishnamurthy, H., Piscitelli, C. L. & Gouaux, E. Unlocking the molecular secrets of sodium-coupled transporters. Nature 459, 347–355 (2009).

    Article  CAS  Google Scholar 

  42. Ilgü, H. et al. Effects of mutations and ligands on the thermostability of the l-arginine/agmatine antiporter AdiC and deduced insights into ligand-binding of human L-type amino acid transporters. Int. J. Mol. Sci. 19, 918 (2018).

    Article  Google Scholar 

  43. Peura, L. et al. Large amino acid transporter 1 (LAT1) prodrugs of valproic acid: new prodrug design ideas for central nervous system delivery. Mol. Pharmacol. 8, 1857–1866 (2011).

    Article  CAS  Google Scholar 

  44. Ylikangas, H. et al. Structure–activity relationship study of compounds binding to large amino acid transporter 1 (LAT1) based on pharmacophore modeling and in situ rat brain perfusion. Eur. J. Pharm. Sci. 48, 523–531 (2013).

    Article  CAS  Google Scholar 

  45. Chien, H.-C. et al. Reevaluating the substrate specificity of the L-type amino acid transporter (LAT1). J. Med. Chem. 61, 7358–7373 (2018).

    Article  CAS  Google Scholar 

  46. Teixeira, S., Grandi, D. S. & Kühn, L. C. Primary structure of the human 4F2 antigen heavy chain predicts a transmembrane protein with a cytoplasmic NH2 terminus. J. Biol. Chem. 262, 9574–9580 (1987).

    CAS  PubMed  Google Scholar 

  47. Hemler, M. E. & Strominger, J. L. Characterization of antigen recognized by the monoclonal antibody (4F2): different molecular forms on human T and B lymphoblastoid cell lines. J. Immunol. 129, 623–628 (1982).

    CAS  PubMed  Google Scholar 

  48. Dickens, D. et al. Modulation of LAT1 (SLC7A5) transporter activity and stability by membrane cholesterol. Sci. Rep. 7, 43580 (2017).

    Article  Google Scholar 

  49. Franca, R., Veljkovic, E., Walter, S., Wagner, C. A. & Verrey, F. Heterodimeric amino acid transporter glycoprotein domains determining functional subunit association. Biochem. J. 388, 435–443 (2005).

    Article  CAS  Google Scholar 

  50. Fenczik, C. A. et al. Distinct domains of CD98hc regulate integrins and amino acid transport. J. Biol. Chem. 276, 8746–8752 (2000).

    Article  Google Scholar 

  51. Wagner, C. A., Lang, F. & Bröer, S. Function and structure of heterodimeric amino acid transporters. Am. J. Physiol. Cell Physiol. 281, 93 (2001).

    Article  Google Scholar 

  52. Bröer, A. et al. Association of 4F2hc with light chains LAT1, LAT2 or y + LAT2 requires different domains. Biochem. J. 355, 725–731 (2001).

    Article  Google Scholar 

  53. Nagamori, S. et al. Novel cystine transporter in renal proximal tubule identified as a missing partner of cystinuria-related plasma membrane protein rBAT/SLC3A1. Proc. Natl Acad. Sci. USA 113, 775–780 (2016).

    Article  CAS  Google Scholar 

  54. Hayes, G. M. et al. Antitumor activity of an anti-CD98 antibody. Int. J. Cancer 137, 710–720 (2015).

    Article  CAS  Google Scholar 

  55. Itoh, K., Inoue, K., Hayashi, H., Suzuki, T. & Masuko, T. Identification of cell proliferation-associated epitope on CD98 oncoprotein using phage display random peptide library. Cancer Sci. 98, 1696–1700 (2007).

    Article  CAS  Google Scholar 

  56. Muramatsu, T. Basigin (CD147), a multifunctional transmembrane glycoprotein with various binding partners. J. Biochem. 159, 481–490 (2016).

    Article  CAS  Google Scholar 

  57. Bajaj, J. et al. CD98-mediated adhesive signaling enables the establishment and propagation of acute myelogenous leukemia. Cancer Cell 30, 792–805 (2016).

    Article  CAS  Google Scholar 

  58. Singh, N. & Ecker, G. F. Insights into the structure, function, and ligand discovery of the large neutral amino acid transporter 1, LAT1. Int. J. Mol. Sci. 19, 1278 (2018).

    Article  Google Scholar 

  59. Drew, D. & Boudker, O. Shared molecular mechanisms of membrane transporters. Annu. Rev. Biochem. 85, 1–30 (2015).

    Google Scholar 

  60. Yan, R., Zhao, X., Lei, J. & Zhou, Q. Structure of the human LAT1–4F2hc heteromeric amino acid transporter complex. Nature 568, 127–130 (2019).

    Article  CAS  Google Scholar 

  61. Goehring, A. et al. Screening and large-scale expression of membrane proteins in mammalian cells for structural studies. Nat. Protoc. 9, 2574–2585 (2014).

    Article  CAS  Google Scholar 

  62. Rothbauer, U. et al. A versatile nanotrap for biochemical and functional studies with fluorescent fusion proteins. Mol. Cell. Proteomics 7, 282–289 (2008).

    Article  CAS  Google Scholar 

  63. Kirchhofer, A. et al. Modulation of protein properties in living cells using nanobodies. Nat. Struct. Mol. Biol. 17, 133–138 (2009).

    Article  Google Scholar 

  64. Geertsma, E. R., Mahmood, N., Schuurman-Wolters, G. K. & Poolman, B. Membrane reconstitution of ABC transporters and assays of translocator function. Nat. Protoc. 3, 256–266 (2008).

    Article  CAS  Google Scholar 

  65. Punjani, A., Rubinstein, J. L., Fleet, D. J. & Brubaker, M. A. cryoSPARC: algorithms for rapid unsupervised cryo-EM structure determination. Nat. Methods 14, 290–296 (2017).

    Article  CAS  Google Scholar 

  66. Mastronarde, D. N. Automated electron microscope tomography using robust prediction of specimen movements. J. Struct. Biol. 152, 36–51 (2005).

    Article  Google Scholar 

  67. Zheng, S. Q. et al. MotionCor2: anisotropic correction of beam-induced motion for improved cryo-electron microscopy. Nat. Methods 14, 331–332 (2017).

    Article  CAS  Google Scholar 

  68. Rohou, A. & Grigorieff, N. CTFFIND4: fast and accurate defocus estimation from electron micrographs. J. Struct. Biol. 192, 216–221 (2015).

    Article  Google Scholar 

  69. Zivanov, J. et al. New tools for automated high-resolution cryo-EM structure determination in RELION-3. Elife 7, e42166 (2018).

    Article  Google Scholar 

  70. Rosenthal, P. B. & Henderson, R. Optimal determination of particle orientation, absolute hand, and contrast loss in single-particle electron cryomicroscopy. J. Mol. Biol. 333, 721–745 (2003).

    Article  CAS  Google Scholar 

  71. Pettersen, E. F. et al. UCSF Chimera—a visualization system for exploratory research and analysis. J. Comput. Chem. 25, 1605–1612 (2004).

    Article  CAS  Google Scholar 

  72. Emsley, P., Lohkamp, B., Scott, W. G. & Cowtan, K. Features and development of Coot. Acta Crystallogr. D 66, 486–501 (2010).

    Article  CAS  Google Scholar 

  73. Emsley, P. & Crispin, M. Structural analysis of glycoproteins: building N-linked glycans with Coot. Acta Crystallogr. D 74, 256–263 (2018).

    Article  CAS  Google Scholar 

  74. Adams, P. D. et al. PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr. D 66, 213–221 (2010).

    Article  CAS  Google Scholar 

  75. Chen, V. B. et al. MolProbity: all-atom structure validation for macromolecular crystallography. Acta Crystallogr. D 66, 12–21 (2010).

    Article  CAS  Google Scholar 

  76. Itoh, K. et al. Phage display cloning and characterization of monoclonal antibody genes and recombinant fab fragment against the CD98 oncoprotein. Jpn. J. Cancer Res. 92, 1313–1321 (2001).

    Article  CAS  Google Scholar 

  77. Brown, A. et al. Tools for macromolecular model building and refinement into electron cryo-microscopy reconstructions. Acta Crystallogr. D 71, 136–153 (2015).

    Article  CAS  Google Scholar 

  78. Wei, L. et al. Specific transport of 3-fluoro-l-α-methyl-tyrosine by LAT1 explains its specificity to malignant tumors in imaging. Cancer Sci. 107, 347–352 (2016).

    Article  CAS  Google Scholar 

  79. Mirshahi, T., Logothetis, D. E. Sassaroli, M. in Ion Channel Localization. Methods in Pharmacology and Toxicology (eds Lopatin, A.N. & Nichols, C.G.) 215–231 (Springer, 2001).

  80. Ohgaki, R. et al. Essential roles of L-type amino acid transporter 1 in syncytiotrophoblast development by presenting fusogenic 4F2hc. Mol. Cell. Biol. 37, e00427-16 (2017).

    Article  Google Scholar 

Download references

Acknowledgements

We thank R. Danev and M. Kikkawa for setting up the cryo-EM infrastructure; K. Ogomori and M. Miyazaki for technical assistance; G. Kasuya, M. Fukuda and R. Taniguchi for discussions; and W. Kühlbrandt for comments on the manuscript. Y.L. was supported by the Toyobo Biotechnology Foundation Fellowship. This work was supported in part by MEXT/JSPS KAKENHI under grant numbers JP16J07405, to Y.L., and JP16H06294, to O.N.; by AMED under grant numbers JP18am0101082, to M.S., JP18gm0810010, to S.N., JP18cm0106131, to Y.K., and JP18am0101115, to O.N.

Author information

Author notes

  1. Yongchan Lee

    Present address: Department of Structural Biology, Max Planck Institute of Biophysics, Frankfurt, Germany

  2. Ryuichiro Ishitani

    Present address: Preferred Networks, Inc., Tokyo, Japan

  3. Takanori Nakane

    Present address: MRC Laboratory of Molecular Biology, Cambridge, UK

Authors and Affiliations

  1. Department of Biological Sciences, Graduate School of Science, The University of Tokyo, Tokyo, Japan

    Yongchan Lee, Tsukasa Kusakizako, Tomohiro Nishizawa, Kazumasa Oda, Ryuichiro Ishitani, Takanori Nakane & Osamu Nureki

  2. Department of Collaborative Research for Bio-Molecular Dynamics, Nara Medical University, Nara, Japan

    Pattama Wiriyasermkul & Shushi Nagamori

  3. Department of Bio-system Pharmacology, Graduate School of Medicine, Osaka University, Osaka, Japan

    Chunhuan Jin, Lili Quan, Ryuichi Ohgaki, Suguru Okuda & Yoshikatsu Kanai

  4. Laboratory for Protein Functional and Structural Biology, RIKEN Center for Biosystems Dynamics Research, Kanagawa, Japan

    Takeshi Yokoyama & Mikako Shirouzu

  5. J-Pharma Co., Ltd, Kanagawa, Japan

    Hitoshi Endou

  6. Global Innovation Center, Shiseido Co., Ltd, Kanagawa, Japan

    Shushi Nagamori

Authors
  1. Yongchan Lee
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Pattama Wiriyasermkul
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Chunhuan Jin
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Lili Quan
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Ryuichi Ohgaki
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Suguru Okuda
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Tsukasa Kusakizako
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Tomohiro Nishizawa
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Kazumasa Oda
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Ryuichiro Ishitani
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. Takeshi Yokoyama
    View author publications

    You can also search for this author in PubMed Google Scholar

  12. Takanori Nakane
    View author publications

    You can also search for this author in PubMed Google Scholar

  13. Mikako Shirouzu
    View author publications

    You can also search for this author in PubMed Google Scholar

  14. Hitoshi Endou
    View author publications

    You can also search for this author in PubMed Google Scholar

  15. Shushi Nagamori
    View author publications

    You can also search for this author in PubMed Google Scholar

  16. Yoshikatsu Kanai
    View author publications

    You can also search for this author in PubMed Google Scholar

  17. Osamu Nureki
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

Y.L. and O.N. conceived the study. Y.L. performed expression screening, purified LAT1–CD98hc and Fab, prepared the cryo-EM samples and determined the structure. Y.L., T.K., T.Ni., R.I., T.Y. and M.S. collected the cryo-EM data. P.W. and S.N. performed the transport assays using proteoliposomes. C.J., L.Q., R.O., S.O. and Y.K. performed the transport assays using X. laevis oocytes. T.Na assisted with high-resolution cryo-EM data processing with RELION. K.O. purified the proteins for biochemical assays. H.E. provided LAT1 inhibitors and assisted with data interpretation. Y.L. and O.N. wrote the manuscript with contribution from all authors. O.N. supervised the project.

Corresponding author

Correspondence to Osamu Nureki.

Ethics declarations

Competing interests

The authors declare no competing interests.

Additional information

Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Integrated supplementary information

Supplementary Figure 1 Purification and biochemical characterization of LAT1-CD98hc.

a, Size-exclusion chromatography profile of LAT1-CD98hc. The fractions used for structural and functional analyses are indicated by a black bar. b, SDS-PAGE analyses of LAT1-CD98hc with or without 20 mM β-ME. c, Chemical structures of LAT1 inhibitors. d, Uptake of l-[3H]Tyr (50 μM, 10 Ci/mol) by purified LAT1-CD98hc reconstituted in liposomes (cyan) or by control liposomes (white). Proteoliposomes preloaded with 4 mM l-Gln (rectangle) exhibited an initial overshoot of uptake (~1–10 min) as compared to those without preloading (circle), indicating the antiport reaction. The proteoliposomes without preloading also slowly accumulated l-[3H]Tyr, suggesting that LAT1 could adopt facilitative transport mode, in addition to the antiport mode. Values are mean ± SEM. n = 3 technical replicates. e, Substrate specificity of LAT1-CD98hc. Proteoliposomes were incubated with various radiolabeled amino acids and the uptake was measured at 10 min. Control liposomes were used for background subtraction. Although we were not able to measure effective uptake of radiolabeled l-Leu and l-Kyn here due to the high background (Source Data), the competitive inhibition assay confirmed the strong inhibition by l-Leu (f), supporting its transport. Values are mean ± SEM. n = 3 technical replicates. f, Inhibition assay. Proteoliposomes were incubated with l-[3H]Tyr (10 μM) in the presence of 30 mM competitive inhibitors in the external solution. Values are mean ± SEM. n = 3 technical replicates. g, Inhibition assay with pre-incubation of 30 μM SKN-102, SKN-103 and JPH203, or 30 mM BCH. h, Tryptophan-fluorescence size exclusion chromatography of LAT1-CD98hc-Fab complexes. I, Representative 2D class averages of LAT1-CD98hc with or without Fab, recorded on a 200 kV JEM2010F microscope equipped with a CCD camera. Fab molecules are highlighted by yellow asterisks. Note that the box sizes are different between the images with or without Fab.

Supplementary Figure 2 Atomic model of LAT1-CD98hc in the cryo-EM density map.

a, Cryo-EM density maps and atomic models are shown for selected regions. TM1–TM12, IL1 and EL2–EL4 of LAT1 (cyan), TM1’ and the linker of CD98hc (green). The disulfide bond, four N-glycans (orange) and five lipids (yellow). b, Density map of LAT1 in different views. Sterol densities are colored yellow. c, Stereo view of TM1a-TM1b and TM6a-TM6b. The EM map shows no prominent density in the putative substrate-binding site, indicating the apo state. The similar view is shown in Fig. 2c.

Supplementary Figure 3 LAT1-CD98hc interaction.

a, Correlation with previous cross-linking data on LAT2-CD98hc37. Upper, cross-linked pairs are shown on the structure as lines. Dotted lines are for a 60% cross-linked pair. Lower, cross-linked pairs in CD98hc and LAT2 are summarized in a table, along with the corresponding residues in LAT1 and the measured Cα-Cα distances. N/A, not applicable because G220 is disordered, and for visualization I219 is used instead. b, Surface electric potential calculation of LAT1 and CD98hc.

Supplementary Figure 4 Topology of LAT1 and its inward-open state.

a, Side view of LAT1. Helices are colored from blue to red. b, Topology diagram of LAT1. The two large triangles indicate the ‘5+5’ inverted repeats. c, Extracellular and cytoplasmic gates. d, Stereo view of the extracellular gate. e, Stereo view of the cytoplasmic gate. f, Comparison of TM1-TM6-TM10 between LAT1, LAT2, ApcT and AdiC. Unique configuration of the TM6a-TM6b loop underlies the specificity of LAT1. g, Sequence comparison of TM1 and TM6 in LAT1, ApcT and AdiC. The position of Gly255 is indicated by an red arrow. Blue boxes indicate the loop between the two discontinuous helices.

Supplementary Figure 5 Transport assays using X. laevis oocytes.

a, Uptake of l-[14C]Leu by X. laevis oocytes co-expressing CD98hc and LAT1 variants measured at four different substrate concentrations. The data used for Fig. 3d are marked by gray dotted rectangles. The specific radioactivities of l-[14C]Leu are 3.3, 3.3, 2.2 and 0.66 Ci/mol for 50, 100, 300 and 1,000 μM, respectively. Values are mean ± SEM. n = 5–9 technical replicates. b, Uptake of l-[14C]Ala. The specific radioactivities of l-[14C]Ala are 5.6, 5.6, 1.9 and 1.1 Ci/mol for 50, 100, 300 and 1,000 μM Ala uptake, respectively. Values are mean ± SEM. n = 4–9 technical replicates. c, Uptake of various radiolabeled amino acids by X. laevis oocytes co-expressing CD98hc and LAT1 (wild type or G255A) measured at two different substrate concentrations. The data from the 10 µM substrate concentrations are used for Fig. 3e. Values are mean ± SEM. n = 6–9 technical replicates. The specific radioactivities used for 100 µM are 2.2, 4.8, 4.5, 3.3, 20 and 1.1 Ci/mol for l-[14C]Trp, l-[14C]Tyr, l-[14C]Phe, l-[14C]Leu, [3H]Val and l-[14C]Ala, respectively. d, Western blotting of CD98hc and LAT1 variants expressed in X. laevis oocytes. Isolated oocyte membranes were fractionated by SDS-PAGE in the absence of DTT, and blotted with anti-LAT1 or anti-CD98hc antibodies. The results confirmed the heterodimer formation for all variants. The data are representative results from two independent experiments. e, Immunofluorescence of X. laevis oocytes, detected by an anti-LAT1 antibody. The results confirmed the plasma membrane localization for all variants.

Supplementary Figure 6 Full-length CD98hc and lipid interactions.

a, Crystal structure of CD98hc-ED (PDB: 2DH3). It superimposes with the present structure with a root mean square deviation (r.m.s.d.) of 1.31 Å for 414 Cα atoms. b, Cryo-EM structure of CD98hc in the LAT1-CD98hc heterodimer. c, Close-up view of the linker. Atom-atom distances are labeled in gray. Linker residues are labeled in red. d, Close-up view of the sterol binding sites. Densities are contoured at 11 σ.

Supplementary Figure 7 Analysis of SLC3-SLC7 interface.

a, The interaction between CD98hc and LAT1. b, TM1'-TM4 interface. c, Linker-Cβ2-Cβ3-Cβ8-EL2 interface. d, Aα8-EL4a and Aα1-Aα2-EL3 interface. e, Sequence alignment of EL2 and TM4. The conserved 9-amino acid stretch of EL2 is enclosed by a blue box. f, Sequence alignment of EL3. g, Sequence alignment of EL4a-EL4b. h, Sequence alignment of CD98hc and rBAT.

Supplementary information

Supplementary Information

Supplementary Figs. 1–7, Supplementary Notes 1 and 2

Reporting Summary

Supplementary Video 1

Cryo-EM density map of LAT1 and CD98hc. The 360°-view of the density map and the atomic model are shown for the LAT1 and CD98hc TMD.

Source Data Fig. 3d,e, Supplementary Fig. 1d–g, Supplementary Fig. 5a–d

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Lee, Y., Wiriyasermkul, P., Jin, C. et al. Cryo-EM structure of the human L-type amino acid transporter 1 in complex with glycoprotein CD98hc. Nat Struct Mol Biol 26, 510–517 (2019). https://doi.org/10.1038/s41594-019-0237-7

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41594-019-0237-7

This article is cited by

Search

Advanced search

Quick links

Nature Briefing: Cancer

Sign up for the Nature Briefing: Cancer newsletter — what matters in cancer research, free to your inbox weekly.

Get what matters in cancer research, free to your inbox weekly. Sign up for Nature Briefing: Cancer