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The solute specificity profiles of nucleobase cation symporter 1 (NCS1) from Zea mays and Setaria viridis illustrate functional flexibility

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Abstract

The solute specificity profiles (transport and binding) for the nucleobase cation symporter 1 (NCS1) proteins, from the closely related C4 grasses Zea mays and Setaria viridis, differ from that of Arabidopsis thaliana and Chlamydomonas reinhardtii NCS1. Solute specificity profiles for NCS1 from Z. mays (ZmNCS1) and S. viridis (SvNCS1) were determined through heterologous complementation studies in NCS1-deficient Saccharomyces cerevisiae strains. The four Viridiplantae NCS1 proteins transport the purines adenine and guanine, but unlike the dicot and algal NCS1, grass NCS1 proteins fail to transport the pyrimidine uracil. Despite the high level of amino acid sequence similarity, ZmNCS1 and SvNCS1 display distinct solute transport and recognition profiles. SvNCS1 transports adenine, guanine, hypoxanthine, cytosine, and allantoin and competitively binds xanthine and uric acid. ZmNCS1 transports adenine, guanine, and cytosine and competitively binds, 5-fluorocytosine, hypoxanthine, xanthine, and uric acid. The differences in grass NCS1 profiles are due to a limited number of amino acid alterations. These amino acid residues do not correspond to amino acids essential for overall solute and cation binding or solute transport, as previously identified in bacterial and fungal NCS1, but rather may represent residues involved in subtle solute discrimination. The data presented here reveal that within Viridiplantae, NCS1 proteins transport a broad range of nucleobase compounds and that the solute specificity profile varies with species.

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Acknowledgments

We thank Regan Huntley and Carol Clark at The Connecticut Agricultural Experiment Station for expert technical assistance. We also thank the Biology Department at the University of Saint Francis, Fort Wayne, Indiana, for use of their confocal microscope. This work was funded by research funds from IPFW to G.S.M. and Hatch Fund CONH00253 to N.P.S.

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The authors declare that they have no competing interests.

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  1. Department of Biology, Indiana University-Purdue University Fort Wayne, 2101 East Coliseum Blvd., Fort Wayne, IN, 46805, USA

    Micah Rapp, Jessica Schein, Kevin A. Hunt, Vamsi Nalam & George S. Mourad

  2. Department of Plant Pathology and Ecology, The Connecticut Agricultural Experiment Station, 123 Huntington St, New Haven, CT, 06511, USA

    Neil P. Schultes

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  1. Micah Rapp
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  2. Jessica Schein
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ESM 1

Alignment of NCS1 proteins from Viridiplantae by ClustalW (Thompson et al. 1994). AcNCS1 Aquilegia coerulea Aquca_009_01069.1; AlNCS1, Arabidopsis lyrata XP_002873103; AtNCS1, Arabidopsis thaliana NP_568122.2; BdNCS1B, Brachypodium distachyon Bradi3g14360.1; BdNCS1A, Brachypodium distachyon Bradi3g51350.1; CarNCS1, Capsella rubella Carubv10003714m; CcNCS1, Citrus clementina Ciclev10011424m; CisNCS1, Citrus sinensis orange1.1g009276m; CrNCS1, Chlamydomonas reinhardtii XP_001694932.1; CsNCS1, Cucumis sativus Cucsa.378830.1; FvNCS1, Fragaria vesca mrna00956.1-v1.0-hybrid; GmNCS1A, Glycine max Glyma13g21131.1; GmNCS1B, Glycine max Glyma10g07230.1i; GrNCS1A, Gossypium raimondii Gorai.006G160100.1; GrNCS1B, Gossypium raimondii Gorai.006G160000.1; HvNCS1, Hordeum vulgare BAJ85216.1; LuNCS1, Linum usitatissimum LuS10014397; MdNCS1, Malus domestica MDP0000253445; MeNCS1, Manihot esculenta cassava4.1_007082m; MgNCS1, Mimulus guttatus mgv1a003893m; MpCCMP1545NCS1, Micromonas pusilla CCMP15451594 MicpuC2.gw1.11.67.1; MpRCC299NCS1, Micromonas pusilla RCC299 55664 EuGene.0200010041; MtNCS1A, Medicago truncatula Medtr7g102810.1; MtNCS1B, Medicago truncatula Medtr1g062130.1; NsNCS1, Nicotiana sylvestris (Schultes & Mourad pers. Comm); ObNCS1, Oryza brachyantha XP_006647657.1; OlNCS1, Ostreococcus lucimarinus 49824|estExt_Genewise_ext.C_Chr_60346; OsNCS1, Oryza sativa LOC_Os02g44680.1; OtNCS1, Ostreococcus tauri Ostta_3|19171|estExt_fgenesh1_pg.C_chrom_06.10330; PgNCS1, Picea glauca gb|BT114389.1; PpNCS1A, Physcomitrella patens Pp1s34_250V6.1 (Phypa_120934); PpNCS1B, Physcomitrella patens Pp1S171_59V6.1 (Phypa_191990); PtNCS1, Populus trichocarpa Potri.006G119500.1; PvNCS1, Phaseolus vulgaris Phvul.007G226300.1; PvNCS1A, Panicum virgatum Pavirv00007850m; PvNCS1B, Panicum virgatum Pavirv00030059m; RcNCS1, Ricinus communis 30078.m002226; SbNCS1, Sorghum bicolor Sb04g032390.1; SiNCS1B, Setaria italica Si016858m; SiNCS1A, Setaria italica Si019168m; SmNCS1, Selaginella moellendorffii gene 93539; StNCS1, Solanum tuberosum XP_006354397; SvNCS1, Setaria viridis AHC53692.1; TcNCS1, Theobroma cacao Thecc1EG022354t1; ThNCS1, Thellungiella halophila Thhalv10015725m; VcNCS1, Volvox carteri Vocar20015267m; VvNCS1, Vitus vinifera GSVIVT01033705001; ZmNCS1, Zea mays GRMZM2G362848_T01. Transmembrane domains for ZmNCS1 are labeled as given in Figure 1 legend. Underlined text in italics represents the predicted chloroplast transit sequence and cleavage site given by ChloroP (Emanuelsson et al. 1999) for ZmNCS1 and SvNCS1. Text in red (with * below) denotes amino acid identity among NCS1 presented, while text in blue (with . below) or green (with : below) denotes strong and weak amino acid similarities, respectively, among NCS1 proteins presented as given by ClustalW. Bold black italic text highlighted with gray background in ZmNCS1 identifies amino acid sequence present in proteomic analysis of maize chloroplast membranes (Friso et al. 2010). The twenty-nine amino acid differences between SvNCS1 and ZmNCS1, past the predicted chloroplast cleavage site of SvNCS1, are as identified in Figure 1 legend. Bold black numbers identify amino acids that differ between SvNCS1 and ZmNCS1 and are not conserved among other NCS1. Bold white numbers with black background identify amino acid differences between SvNCS1 and ZmNCS1 but are predominantly conserved among all other NCS1 presented. Text in the AtNCS1 sequence with aqua background identifies amino acid sites mutated by site-directed mutagenesis (Witz et al. 2014) (DOCX 59 kb)

ESM 2

Time course for uptake of radiolabeled nucleobase of SvNCS1 and ZmNCS1 in S. cerevisiae. Uptake of 1.0 μM [8-3H]-hypoxanthine in S. cerevisiae strains containing SvNCS1 (pCC207) over time (a), of 0.5 μM [2,8-3H]-adenine in S. cerevisiae strain (fcy2∆) containing SvNCS1 (pCC207) (b) or ZmNCS1 (pNS487) (c) over time (GIF 46 kb)

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Rapp, M., Schein, J., Hunt, K.A. et al. The solute specificity profiles of nucleobase cation symporter 1 (NCS1) from Zea mays and Setaria viridis illustrate functional flexibility. Protoplasma 253, 611–623 (2016). https://doi.org/10.1007/s00709-015-0838-x

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