Cysteine 467 of the ASCT2 Amino Acid Transporter Is a Molecular Determinant of the Antiport Mechanism
Abstract
:1. Introduction
2. Results
2.1. Reconstitution of C467A Mutant in Proteoliposomes
2.2. Functional Characterization of C467A Unidirectional Transport in Proteoliposomes
2.3. Kinetic Characterization of C467A Unidirectional Transport in Proteoliposomes
2.4. In Silico Analysis of hASCT2 Transport Cycle
3. Discussion
4. Materials and Methods
4.1. Recombinant Production of hASCT2 WT and C467A Mutant
4.2. Solubilization and Purification of hASCT2 WT and C467A Mutant
4.3. Inclusion of Cholesterol in Liposome Preparation
4.4. Reconstitution of the Recombinant hASCT2 into Liposome
4.5. Transport Measurements
4.6. Data Analysis
4.7. Other Methods
Author Contributions
Funding
Data Availability Statement
Conflicts of Interest
References
- Scalise, M.; Pochini, L.; Console, L.; Losso, M.A.; Indiveri, C. The Human SLC1A5 (ASCT2) Amino Acid Transporter: From Function to Structure and Role in Cell Biology. Front. Cell Dev. Biol. 2018, 6, 96. [Google Scholar] [CrossRef] [PubMed]
- Newsholme, P.; Procopio, J.; Lima, M.M.; Pithon-Curi, T.C.; Curi, R. Glutamine and glutamate—Their central role in cell metabolism and function. Cell Biochem. Funct. 2003, 21, 1–9. [Google Scholar] [CrossRef] [PubMed]
- Bhutia, Y.D.; Ganapathy, V. Glutamine transporters in mammalian cells and their functions in physiology and cancer. Biochim. Biophys. Acta 2016, 1863, 2531–2539. [Google Scholar] [CrossRef] [PubMed]
- Kanai, Y.; Clemencon, B.; Simonin, A.; Leuenberger, M.; Lochner, M.; Weisstanner, M.; Hediger, M.A. The SLC1 high-affinity glutamate and neutral amino acid transporter family. Mol. Aspects Med. 2013, 34, 108–120. [Google Scholar] [CrossRef]
- Kanai, Y.; Hediger, M.A. The glutamate/neutral amino acid transporter family SLC1: Molecular, physiological and pharmacological aspects. Pflugers Arch. 2004, 447, 469–479. [Google Scholar] [CrossRef]
- Freidman, N.; Chen, I.; Wu, Q.; Briot, C.; Holst, J.; Font, J.; Vandenberg, R.; Ryan, R. Amino Acid Transporters and Exchangers from the SLC1A Family: Structure, Mechanism and Roles in Physiology and Cancer. Neurochem. Res. 2020, 45, 1268–1286. [Google Scholar] [CrossRef]
- Scalise, M.; Console, L.; Cosco, J.; Pochini, L.; Galluccio, M.; Indiveri, C. ASCT1 and ASCT2: Brother and Sister? SLAS Discov. 2021, 26, 1148–1163. [Google Scholar] [CrossRef]
- Canul-Tec, J.C.; Kumar, A.; Dhenin, J.; Assal, R.; Legrand, P.; Rey, M.; Chamot-Rooke, J.; Reyes, N. The ion-coupling mechanism of human excitatory amino acid transporters. EMBO J. 2021, 41, e108341. [Google Scholar] [CrossRef]
- Arriza, J.L.; Kavanaugh, M.P.; Fairman, W.A.; Wu, Y.N.; Murdoch, G.H.; North, R.A.; Amara, S.G. Cloning and expression of a human neutral amino acid transporter with structural similarity to the glutamate transporter gene family. J. Biol. Chem. 1993, 268, 15329–15332. [Google Scholar] [CrossRef]
- Yamamoto, T.; Nishizaki, I.; Furuya, S.; Hirabayashi, Y.; Takahashi, K.; Okuyama, S.; Yamamoto, H. Characterization of rapid and high-affinity uptake of L-serine in neurons and astrocytes in primary culture. FEBS Lett. 2003, 548, 69–73. [Google Scholar] [CrossRef] [Green Version]
- Pochini, L.; Scalise, M.; Galluccio, M.; Indiveri, C. Membrane transporters for the special amino acid glutamine: Structure/function relationships and relevance to human health. Front. Chem. 2014, 2, 61. [Google Scholar] [CrossRef] [Green Version]
- Scalise, M.; Pochini, L.; Pingitore, P.; Hedfalk, K.; Indiveri, C. Cysteine is not a substrate but a specific modulator of human ASCT2 (SLC1A5) transporter. FEBS Lett. 2015, 589, 3617–3623. [Google Scholar] [CrossRef]
- Utsunomiya-Tate, N.; Endou, H.; Kanai, Y. Cloning and functional characterization of a system ASC-like Na+-dependent neutral amino acid transporter. J. Biol. Chem. 1996, 271, 14883–14890. [Google Scholar] [CrossRef] [Green Version]
- Torres-Zamorano, V.; Leibach, F.H.; Ganapathy, V. Sodium-dependent homo- and hetero-exchange of neutral amino acids mediated by the amino acid transporter ATB degree. Biochem. Biophys. Res. Commun. 1998, 245, 824–829. [Google Scholar] [CrossRef]
- Pingitore, P.; Pochini, L.; Scalise, M.; Galluccio, M.; Hedfalk, K.; Indiveri, C. Large scale production of the active human ASCT2 (SLC1A5) transporter in Pichia pastoris—Functional and kinetic asymmetry revealed in proteoliposomes. Biochim. Biophys. Acta 2013, 1828, 2238–2246. [Google Scholar] [CrossRef] [Green Version]
- Scalise, M.; Pochini, L.; Cosco, J.; Aloe, E.; Mazza, T.; Console, L.; Esposito, A.; Indiveri, C. Interaction of Cholesterol With the Human SLC1A5 (ASCT2): Insights Into Structure/Function Relationships. Front. Mol. Biosci. 2019, 6, 110. [Google Scholar] [CrossRef] [Green Version]
- Scalise, M.; Pochini, L.; Panni, S.; Pingitore, P.; Hedfalk, K.; Indiveri, C. Transport mechanism and regulatory properties of the human amino acid transporter ASCT2 (SLC1A5). Amino Acids 2014, 46, 2463–2475. [Google Scholar] [CrossRef]
- Mazza, T.; Scalise, M.; Pappacoda, G.; Pochini, L.; Indiveri, C. The involvement of sodium in the function of the human amino acid transporter ASCT2. FEBS Lett. 2021, 595, 3030–3041. [Google Scholar] [CrossRef]
- Qiu, B.; Matthies, D.; Fortea, E.; Yu, Z.; Boudker, O. Cryo-EM structures of excitatory amino acid transporter 3 visualize coupled substrate, sodium, and proton binding and transport. Sci. Adv. 2021, 7. [Google Scholar] [CrossRef]
- Boudker, O.; Ryan, R.M.; Yernool, D.; Shimamoto, K.; Gouaux, E. Coupling substrate and ion binding to extracellular gate of a sodium-dependent aspartate transporter. Nature 2007, 445, 387–393. [Google Scholar] [CrossRef]
- Parker, J.L.; Deme, J.C.; Kolokouris, D.; Kuteyi, G.; Biggin, P.C.; Lea, S.M.; Newstead, S. Molecular basis for redox control by the human cystine/glutamate antiporter system xc. Nat. Commun. 2021, 12, 7147. [Google Scholar] [CrossRef]
- Errasti-Murugarren, E.; Fort, J.; Bartoccioni, P.; Diaz, L.; Pardon, E.; Carpena, X.; Espino-Guarch, M.; Zorzano, A.; Ziegler, C.; Steyaert, J.; et al. L amino acid transporter structure and molecular bases for the asymmetry of substrate interaction. Nat. Commun. 2019, 10, 1807. [Google Scholar] [CrossRef] [Green Version]
- Nicolas-Arago, A.; Fort, J.; Palacin, M.; Errasti-Murugarren, E. Rush Hour of LATs towards Their Transport Cycle. Membranes 2021, 11, 602. [Google Scholar] [CrossRef]
- Pavic, A.; Holmes, A.O.M.; Postis, V.L.G.; Goldman, A. Glutamate transporters: A broad review of the most recent archaeal and human structures. Biochem. Soc. Trans. 2019, 47, 1197–1207. [Google Scholar] [CrossRef] [Green Version]
- Canul-Tec, J.C.; Assal, R.; Cirri, E.; Legrand, P.; Brier, S.; Chamot-Rooke, J.; Reyes, N. Structure and allosteric inhibition of excitatory amino acid transporter 1. Nature 2017, 544, 446–451. [Google Scholar] [CrossRef]
- Garaeva, A.A.; Oostergetel, G.T.; Gati, C.; Guskov, A.; Paulino, C.; Slotboom, D.J. Cryo-EM structure of the human neutral amino acid transporter ASCT2. Nat. Struct. Mol. Biol. 2018, 25, 515–521. [Google Scholar] [CrossRef]
- Scalise, M.; Pochini, L.; Console, L.; Pappacoda, G.; Pingitore, P.; Hedfalk, K.; Indiveri, C. Cys Site-Directed Mutagenesis of the Human SLC1A5 (ASCT2) Transporter: Structure/Function Relationships and Crucial Role of Cys467 for Redox Sensing and Glutamine Transport. Int. J. Mol. Sci 2018, 19, 648. [Google Scholar] [CrossRef] [Green Version]
- Scalise, M.; Mazza, T.; Pappacoda, G.; Pochini, L.; Cosco, J.; Rovella, F.; Indiveri, C. The Human SLC1A5 Neutral Amino Acid Transporter Catalyzes a pH-Dependent Glutamate/Glutamine Antiport, as Well. Front. Cell Dev. Biol 2020, 8, 603. [Google Scholar] [CrossRef]
- Mazmanian, K.; Sargsyan, K.; Grauffel, C.; Dudev, T.; Lim, C. Preferred Hydrogen-Bonding Partners of Cysteine: Implications for Regulating Cys Functions. J. Phys. Chem. B 2016, 120, 10288–10296. [Google Scholar] [CrossRef]
- Garaeva, A.A.; Guskov, A.; Slotboom, D.J.; Paulino, C. A one-gate elevator mechanism for the human neutral amino acid transporter ASCT2. Nat. Commun. 2019, 10, 3427. [Google Scholar] [CrossRef] [Green Version]
- Oberg, F.; Sjohamn, J.; Conner, M.T.; Bill, R.M.; Hedfalk, K. Improving recombinant eukaryotic membrane protein yields in Pichia pastoris: The importance of codon optimization and clone selection. Mol. Membr. Biol. 2011, 28, 398–411. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Hanson, M.A.; Cherezov, V.; Griffith, M.T.; Roth, C.B.; Jaakola, V.P.; Chien, E.Y.; Velasquez, J.; Kuhn, P.; Stevens, R.C. A specific cholesterol binding site is established by the 2.8 A structure of the human beta2-adrenergic receptor. Structure 2008, 16, 897–905. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Palmieri, F.; Klingenberg, M. Direct methods for measuring metabolite transport and distribution in mitochondria. Methods Enzymol. 1979, 56, 279–301. [Google Scholar] [CrossRef] [PubMed]
- Pettersen, E.F.; Goddard, T.D.; Huang, C.C.; Couch, G.S.; Greenblatt, D.M.; Meng, E.C.; Ferrin, T.E. UCSF Chimera—A visualization system for exploratory research and analysis. J. Comput. Chem. 2004, 25, 1605–1612. [Google Scholar] [CrossRef] [Green Version]
Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. |
© 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).
Share and Cite
Scalise, M.; Pappacoda, G.; Mazza, T.; Console, L.; Pochini, L.; Indiveri, C. Cysteine 467 of the ASCT2 Amino Acid Transporter Is a Molecular Determinant of the Antiport Mechanism. Int. J. Mol. Sci. 2022, 23, 1127. https://doi.org/10.3390/ijms23031127
Scalise M, Pappacoda G, Mazza T, Console L, Pochini L, Indiveri C. Cysteine 467 of the ASCT2 Amino Acid Transporter Is a Molecular Determinant of the Antiport Mechanism. International Journal of Molecular Sciences. 2022; 23(3):1127. https://doi.org/10.3390/ijms23031127
Chicago/Turabian StyleScalise, Mariafrancesca, Gilda Pappacoda, Tiziano Mazza, Lara Console, Lorena Pochini, and Cesare Indiveri. 2022. "Cysteine 467 of the ASCT2 Amino Acid Transporter Is a Molecular Determinant of the Antiport Mechanism" International Journal of Molecular Sciences 23, no. 3: 1127. https://doi.org/10.3390/ijms23031127