Structure of eukaryotic purine/H+ symporter UapA suggests a role for homodimerization in transport activity

Nature Communications

Volumn 7, Issue , 2016, Pages

  Alguel, Yilmaz ^a   Amillis, Sotiris ^b   Leung, James ^a   Lambrinidis, George ^b   Capaldi, Stefano ^a,c   Scull, Nicola J ^a   Craven, Gregory ^a   Iwata, So ^a,d   Armstrong, Alan ^a   Mikros, Emmanuel ^b   Diallinas, George ^b   Cameron, Alexander D ^e   Byrne, Bernadette ^a  

^a IMPERIAL COLLEGE LONDON   (United Kingdom)

^b UNIVERSITY OF ATHENS   (Greece)

^c UNIVERSITY OF VERONA   (Italy)

^d JAPAN SCIENCE AND TECHNOLOGY AGENCY   (Japan)

^e UNIVERSITY OF WARWICK   (United Kingdom)

Author keywords

[No Author keywords available]

Indexed keywords

BACTERIAL PROTEIN; PRESTIN; UAPA PROTEIN; UNCLASSIFIED DRUG; URAA PROTEIN; URACIL; URATE TRANSPORTER; XANTHINE; CARRIER PROTEIN; FUNGAL PROTEIN; PROTON; RECOMBINANT PROTEIN; UAPA PROTEIN, ASPERGILLUS NIDULANS;

AMINO ACID; CRYSTAL STRUCTURE; EUKARYOTE; FUNGUS; ORGANIC COMPOUND; PIGMENT;

AMINO TERMINAL SEQUENCE; ANION EXCHANGE; ARTICLE; ASPERGILLUS NIDULANS; BINDING AFFINITY; BINDING SITE; CELL MEMBRANE; CELLULAR DISTRIBUTION; COMPLEX FORMATION; CONFORMATIONAL TRANSITION; CRYSTAL STRUCTURE; CRYSTALLIZATION; DIMERIZATION; DISULFIDE BOND; ENZYME SPECIFICITY; ENZYME SUBSTRATE COMPLEX; HETEROLOGOUS EXPRESSION; HOMODIMERIZATION; MOLECULAR DYNAMICS; NONHUMAN; PROTEIN BINDING; PROTEIN CONFORMATION; PROTEIN DOMAIN; PROTEIN PROTEIN INTERACTION; PROTEIN STRUCTURE; PROTEIN TRANSPORT; STRUCTURE ACTIVITY RELATION; CHEMISTRY; GENE EXPRESSION; GENETICS; KINETICS; METABOLISM; MOLECULAR MODEL; MUTATION; PROTEIN MULTIMERIZATION; PROTEIN SECONDARY STRUCTURE; PROTEIN TERTIARY STRUCTURE; SACCHAROMYCES CEREVISIAE; THERMODYNAMICS; TRANSPORT AT THE CELLULAR LEVEL; X RAY CRYSTALLOGRAPHY;

EMERICELLA NIDULANS; EUKARYOTA; FUNGI;

ASPERGILLUS NIDULANS; BIOLOGICAL TRANSPORT; CRYSTALLOGRAPHY, X-RAY; FUNGAL PROTEINS; GENE EXPRESSION; KINETICS; MEMBRANE TRANSPORT PROTEINS; MODELS, MOLECULAR; MUTATION; PROTEIN MULTIMERIZATION; PROTEIN STRUCTURE, SECONDARY; PROTEIN STRUCTURE, TERTIARY; PROTONS; RECOMBINANT PROTEINS; SACCHAROMYCES CEREVISIAE; SUBSTRATE SPECIFICITY; THERMODYNAMICS; XANTHINE;

EID: 84973387903     PISSN: None     EISSN: 20411723     Source Type: Journal    
DOI: 10.1038/ncomms11336     Document Type: Article

References (56)

1. Gournas, C., Papageorgiou, I. & Diallinas, G. The nucleobase-ascorbate transporter (NAT) family: genomics, evolution, structure-function relationships and physiological role. Mol. BioSyst. 4, 404 (2008).
2. Diallinas, G. & Gournas, C. The ubiquitous Nucleobase-Ascorbate Transporter (NAT) family: lessons from model microbial genetic systems. Channels 2, 363-372 (2008).
3. Tsukaguchi, H. et al. A family of mammalian Na+-dependent L-ascorbic acid transporters. Nature 399, 70-75 (1999).
4. Goudela, S., Reichard, U., Amillis, S. & Diallinas, G. Characterization and kinetics of the major purine transporters in Aspergillus fumigatus. Fungal Genet. Biol. 45, 459-472 (2008).
5. Yamamoto, S. et al. Identification and functional characterization of the first nucleobase transporter in mammals: implication in the species difference in the intestinal absorption mechanism of nucleobases and their analogs between higher primates and other mammals. J. Biol. Chem. 285, 6522-6531 (2010).
6. Diallinas, G. & Scazzocchio, C. A gene coding for the uric acid-xanthine permease of Aspergillus nidulans: inactivational cloning, characterization, and sequence of a cis-acting mutation. Genetics 122, 341-350 (1989).
7. Papageorgiou, I. et al. Specific interdomain synergy in the UapA transporter determines its unique specificity for uric acid among NAT carriers. J. Mol. Biol. 382, 1121-1135 (2008).
8. Kosti, V., Papageorgiou, I. & Diallinas, G. Dynamic elements at both cytoplasmically and extracellularly facing sides of the uapa transporter selectively control the accessibility of substrates to their translocation pathway. J. Mol. Biol. 397, 1132-1143 (2010).
9. Kosti, V., Lambrinidis, G., Myrianthopoulos, V., Diallinas, G. & Mikros, E. Identification of the substrate recognition and transport pathway in a eukaryotic member of the nucleobase-ascorbate transporter (NAT) family. PLoS ONE 7, e41939 (2012).
10. Lu, F. et al. Structure and mechanism of the uracil transporter UraA. Nature 472, 243-246 (2011).
11. Martzoukou, O. et al. Oligomerization of the UapA purine transporter is critical for ER-exit, plasma membrane localization and turnover. J. Mol. Biol. 427, 2679-2696 (2015).
12. Torres, G. E. et al. Oligomerization and trafficking of the human dopamine transporter. Mutational analysis identifies critical domains important for the functional expression of the transporter. J. Biol. Chem. 278, 2731-2739 (2003).
13. Kilic, F. & Rudnick, G. Oligomerization of serotonin transporter and its functional consequences. Proc. Natl Acad. Sci. USA 97, 3106-3111 (2000).
14. De Zutter, J. K., Levine, K. B., Deng, D. & Carruthers, A. Sequence determinants of GLUT1 oligomerization: analysis by homology-scanning mutagenesis. J. Biol. Chem. 288, 20734-20744 (2013).
15. Zhen, J. et al. Dopamine transporter oligomerization: impact of combining protomers with differential cocaine analog binding affinities. J. Neurochem. 133, 167-173 (2015).
16. Tao, Y. et al. Structure of a eukaryotic SWEET transporter in a homotrimeric complex. Nature 527, 259-263 (2015).
17. Leung, J., Cameron, A. D., Diallinas, G. & Byrne, B. Stabilizing the heterologously expressed uric acid-xanthine transporter UapA from the lower eukaryote Aspergillus nidulans. Mol. Membr. Biol. 30, 32-42 (2013).
18. Leung, J., Karachaliou, M., Alves, C., Diallinas, G. & Byrne, B. Expression and purification of a functional uric acid-xanthine transporter (UapA). Protein Expr. Purif. 72, 139-146 (2010).
19. Koukaki, M. et al. The nucleobase-ascorbate transporter (NAT) signature motif in UapA defines the function of the purine translocation pathway. J. Mol. Biol. 350, 499-513 (2005).
20. Wang, J. B., Moriwaki, A. & Uhl, G. R. Dopamine transporter cysteine mutants: second extracellular loop cysteines are required for transporter expression. J. Neurochem. 64, 1416-1419 (1995).
21. Chen, J. G., Liu-Chen, S. & Rudnick, G. External cysteine residues in the serotonin transporter. Biochemistry 36, 1479-1486 (1997).
22. Diallinas, G. Understanding transporter specificity and the discrete appearance of channel-like gating domains in transporters. Front. Pharmacol. 5, 207 (2014).
23. Geertsma, E. R. et al. Structure of a prokaryotic fumarate transporter reveals the architecture of the SLC26 family. Nature Struct. Mol. Biol. 22, 803-808 (2015).
24. Arakawa, T. et al. Crystal structure of the anion exchanger domain of human erythrocyte band 3. Science 350, 680-684 (2015).
25. 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 445, 387-393 (2007).
26. Zhou, X. et al. Structural basis of the alternating-access mechanism in a bile acid transporter. Nature 505, 569-573 (2013).
27. Luo, P. et al. Crystal structure of a phosphorylation-coupled vitamin C transporter. Nature Struct. Mol. Biol. 22, 238-241 (2015).
28. Lee, C. et al. A two-domain elevator mechanism for sodium/proton antiport. Nature 501, 573-577 (2013).
29. Coincon, M. et al. Crystal structures reveal the molecular basis of ion translocation in sodium/proton antiporters. Nature Struct. Mol. Biol. 23, 248-255 (2016).
30. Xuan, Y. H. et al. Functional role of oligomerization for bacterial and plant SWEET sugar transporter family. Proc. Natl Acad. Sci. USA 110, E3685-E3694 (2013).
31. Shi, Y. Common folds and transport mechanisms of secondary active transporters. Annu. Rev. Biophys. 42, 51-72 (2013).
32. Drew, D. et al. GFP-based optimization scheme for the overexpression and purification of eukaryotic membrane proteins in Saccharomyces cerevisiae. Nat. Protoc. 3, 784-798 (2008).
33. Woolford, C. A. et al. The PEP4 gene encodes an aspartyl protease implicated in the posttranslational regulation of Saccharomyces cerevisiae vacuolar hydrolases. Mol. Cell Biol. 6, 2500-2510 (2010).
34. Kabsch, W. XDS. Acta Crystallogr. D Biol. Crystallogr. 66, 125-132 (2010).
35. Winter, G. Xia2: an expert system for macromolecular crystallography data reduction. J. Appl. Crystallogr. 43, 186-190 (2010).
36. Collaborative Computational Project, Number 4. The CCP4 suite: programs for protein crystallography. Acta Crystallogr. D Biol. Crystallogr. 50, 760-763 (1994).
37. Sheldrick, G. M. Experimental phasing with SHELXC/D/E: combining chain tracing with density modification. Acta Crystallogr. D Biol. Crystallogr. 66, 479-485 (2010).
38. Bricogne, G., Vonrhein, C., Flensburg, C., Schiltz, M. & Paciorek, W. Generation, representation and flow of phase information in structure determination: recent developments in and around SHARP 2.0. Acta Crystallogr. D Biol. Crystallogr. 59, 2023-2030 (2003).
39. Cowtan, K. 'dm': an automated procedure for phase improvement by density modification. Joint CCP4 ESF-EACBM Newslett. Prot. Crystallogr. 31, 34-38 (1994).
40. Emsley, P. & Cowtan, K. Coot: model-building tools for molecular graphics. Acta Crystallogr. D Biol. Crystallogr. 60, 2126-2132 (2004).
41. Jones, T. A. & Kjeldgaard, M. Electron-density map interpretation. Meth. Enzymol. 277, 173-208 (1997).
42. Adams, P. D. et al. PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr. D Biol. Crystallogr. 66, 213-221 (2010).
43. Schröder, G. F., Levitt, M. & Brunger, A. T. Super-resolution biomolecular crystallography with low-resolution data. Nature 464, 1218-1222 (2010).
44. Kleywegt, G. J., Zou, J. Y., Kjeldgaard, M., Jones, T. A. & Around, O. in Volume F: Crystallography of Biological Macromolecules. (eds Rossmann, M. G. & Arnold, E.) (Kluwer, 2001).
45. Winn, M. D., Isupov, M. N. & Murshudov, G. N. Use of TLS parameters to model anisotropic displacements in macromolecular refinement. Acta Crystallogr. D Biol. Crystallogr. 57, 122-133 (2001).
46. DeLaBarre, B. & Brunger, A. T. Considerations for the refinement of low-resolution crystal structures. Acta Crystallogr. D Biol. Crystallogr. 62, 923-932 (2006).
47. Dimaio, F. et al. Improved low-resolution crystallographic refinement with Phenix and Rosetta. Nat. Methods 10, 1102-1104 (2013).
48. Davis, I. W. et al. MolProbity: all-atom contacts and structure validation for proteins and nucleic acids. Nucleic Acids Res. 35, W375-W383 (2007).
49. Kleywegt, G. J. & Jones, T. A. A super position. ESF/CCP4 Newslett. 31, 9-14 (1994).
50. DeLano, W. The PyMOL Molecular Graphics System. Available at pymol.org, Schrödinger, LLC (2002).
51. Pantazopoulou, A. et al. Differential physiological and developmental expression of the UapA and AzgA purine transporters in Aspergillus nidulans. Fungal Genet. Biol. 44, 627-640 (2007).
52. Koukaki, M., Giannoutsou, E., Karagouni, A. & Diallinas, G. A novel improved method for Aspergillus nidulans transformation. J. Microbiol. Methods 55, 687-695 (2003).
53. Karachaliou, M. et al. The arrestin-like protein ArtA is essential for ubiquitination and endocytosis of the UapA transporter in response to both broad-range and specific signals. Mol. Microbiol. 88, 301-317 (2013).
54. Krypotou, E. & Diallinas, G. Transport assays in filamentous fungi: kinetic characterization of the UapC purine transporter of Aspergillus nidulans. Fungal Genet. Biol. 63, 1-8 (2014).
55. Bowers, K. J. et al. in Proc. 2006 ACM/IEEE Conf. Supercomput. (SC06) (ACM, 2006).
56. Gournas, C., Amillis, S., Vlanti, A. & Diallinas, G. Transport-dependent endocytosis and turnover of a uric acid-xanthine permease. Mol. Microbiol. 75, 246-260 (2010).