XB-ART-53078Elife. March 3, 2017; 6
Origin and evolution of transporter substrate specificity within the NPF family.
Despite vast diversity in metabolites and the matching substrate specificity of their transporters, little is known about how evolution of transporter substrate specificities is linked to emergence of substrates via evolution of biosynthetic pathways. Transporter specificity towards the recently evolved glucosinolates characteristic of Brassicales is shown to evolve prior to emergence of glucosinolate biosynthesis. Furthermore, we show that glucosinolate transporters belonging to the ubiquitous NRT1/PTR FAMILY (NPF) likely evolved from transporters of the ancestral cyanogenic glucosides found across more than 2500 species outside of the Brassicales. Biochemical characterization of orthologs along the phylogenetic lineage from cassava to A. thaliana, suggests that alterations in the electrogenicity of the transporters accompanied changes in substrate specificity. Linking the evolutionary path of transporter substrate specificities to that of the biosynthetic pathways, exemplify how transporter substrate specificities originate and evolve as new biosynthesis pathways emerge.
PubMed ID: 28257001
PMC ID: PMC5336358
Article link: Elife.
Genes referenced: ag1 rragc
Article Images: [+] show captions
|Figure 2. Biochemical characterization of the indole-specific glucosinolate transporter GTR3.(A–B) Normalized IV (Current-Voltage) curve of 4MTB (A)- and I3M (B)-induced currents for GTR1 (black circles)- and GTR3 (green circles)-expressing oocytes exposed to 100 µM substrate at pH5. Both GTR1 and GTR3 currents were normalized to GTR1 currents elicited at saturating 4MTB concentrations and at a membrane potential of −60 mV (Error bars represent ± s.e., n = 6, experiment repeated two times). (C–D) Time-dependent accumulation of I3M (C) and 4MTB (D), respectively, relative to assay media concentration in GTR1- and GTR3-expressing oocytes. Accumulated 4MTB or I3M were quantified by LC-MS in 3 × 5 oocytes for each gene after 3, 4 and 5 hr of incubation in a standard pH5 Kulori buffer containing 0.2 mM I3M or 0.2 mM 4MTB (error bars represent ± s.d. n = 3). Dotted line represents media concentration. (E–F) Normalized I3M-induced currents for GTR3 (E) or GTR1 (F) measured at a membrane potential of −60 mV and pH 5 plotted against increasing I3M concentrations. The saturation curve was fitted with a Michaelis-Menten equation represented by a solid line. Each oocyte dataset was normalized to currents elicited at 0.8 mM I3M concentration at −60 mV. The insert shows the apparent Km as a function of membrane potential. Error bars represent ± s.e. of mean, n = 6, experiment repeated two times.DOI: http://dx.doi.org/10.7554/eLife.19466.005Figure 2—figure supplement 1. Uptake of 4MTB and I3M by GTR1 and GTR3 expressed in X. laevis oocytes.(A–B) Time-dependent I3M (A) and 4MTB (B) uptake by GTR1- and GTR3-expressing oocytes. This data was shown in Figure 2 as up-concentration relative to assay media concentration. Here substrate amounts are shown in pmol per oocyte. Accumulated 4MTB or I3M was quantified by LC-MS in 3 × 5 oocytes for each gene after 3, 4 and 5 hr of incubation in a standard pH5 kulori buffer containing 0.2M I3M or 0.2 µM 4MTB. Error bars represent ± s.d. of mean for data, n = 3.DOI: http://dx.doi.org/10.7554/eLife.19466.006|
|Figure 2—figure supplement 2. GTR2 indole glucosinolate Km measurement.Normalized I3M-induced currents of GTR2-expressing oocytes measured at a membrane potential of −60 mV and pH 5 were plotted against increasing I3M concentrations. The saturation curve was fitted with a Michaelis-Menten equation – represented by a solid line. Insert shows apparent Km as a function of clamped membrane potential. Each oocyte dataset was normalized to I3M-induced currents elicited at 0.8 mM I3M concentration at −60 mV. The insert shows the apparent Km as a function of membrane potential. Error bars represent ± s.e. of mean for data obtained from six different oocytes per experiment, experiment repeated two times.DOI: http://dx.doi.org/10.7554/eLife.19466.007|
|Figure 3. Substrate competition assays of GTRs in X. laevis oocytes.(A–D) Competition for uptake of I3M and 4MTB into oocytes expressing GTR1 or GTR3. (A) Quantification of 4MTB uptake when oocytes were exposed to high 4MTB concentration (2 mM) alone or in combination with low concentration of I3M (0.2 mM). (B) Quantification of I3M uptake when low I3M concentration (0.2 mM) was competed with high concentration of 4MTB (2 mM). (C) Quantification of I3M when oocytes were exposed to high I3M concentration (2 mM) alone or in combination with low concentration of 4MTB (0.2 mM). (D) Quantification of 4MTB uptake when oocytes were exposed to low I3M concentration (0.2 mM) alone or in combination with high concentration of 4MTB (2 mM). Accumulated 4MTB (A and D) or I3M (B and C) was quantified by LC-MS in 3 × 5 oocytes for each gene. Two tailed T-test, **p<0.001 vs non-competed, *p<0.05 vs non-competed. NS= not significantly different (Error bars represent ± s.d. of mean for data obtained from three times five different oocytes per experiment). (E–G) Quantification of nitrate and glucosinolate competition assays. (E) Quantification of I3M uptake in GTR3-expressing oocytes when saturating I3M concentration (0.1 mM) is competed with high concentration of NO3- (10 mM) or saturating concentration of 4MTB (0.1 mM). (F) Quantification of 4MTB uptake in GTR3-expressing oocytes when saturating 4MTB concentration (0.1 mM) is competed with high concentration of NO3- (10 mM). (G) Quantification of NO3- uptake in GTR3-expressing oocytes when high concentration of NO3- (10 mM) is competed by 0.1 mM 4MTB or saturating concentration of I3M (0.1 mM). Accumulated I3M (E) or 4MTB (F) was quantified by LC-MS in 3 × 5 oocytes for each gene. Accumulated NO3- (G) was quantified by ICP-MS in three oocytes for each gene. Error bars represent ± s.d. of mean, n = 3. Groups in subfigures are determined by one-way ANOVA followed by Holm-Sidak post-hoc analysis (p<0.05).DOI: http://dx.doi.org/10.7554/eLife.19466.008|
|Figure 4—figure supplement 3. Validation of gtr3 T-DNA insertion mutants.Genomic location of T-DNA insertion in gtr3. Arrows indicate location of RT-PCR primers. RT-PCR analysis on RNA isolated from wild type Col-0, gtr3, gtr1 gtr2, gtr1gtr2 gtr3 using GTR3-specific primers. TUB1 (AT1G75780)-specific primers were used as internal control. See Supplementary Materials and methods for primer sequences.DOI: http://dx.doi.org/10.7554/eLife.19466.016|
|Figure 5—figure supplement 2. TEVC electrophysiology measurements of AtGTR1, AtGTR3 and Me15G74000.(A–D) 4MTB (black circles)- and I3M (green circles)-induced currents in oocytes expressing GTR homologs that showed glucosinolate uptake in Figure 5B. Expressing and non-expressing oocytes were exposed to 0.2 mM 4MTB or I3M and induced currents were measured at membrane potentials between 0 mV and −180 mV in 20 mV increments at pH5 (Error bars represent ± s.d. of mean for data obtained from four different oocytes per experiment, experiment repeated two times).DOI: http://dx.doi.org/10.7554/eLife.19466.019|
|Figure 5—figure supplement 3. Expression analysis of YFP-tagged (C-terminal) GTRs and GTR homologs from B. rapa, C. papaya, T. cacao and cassava in X. laevis oocytes.YFP fluorescence in yellow (top) and the plasmamembrane-staining dye FM4-64fx in blue (bottom). Shown are representative images of at least three oocytes.DOI: http://dx.doi.org/10.7554/eLife.19466.020|
|Figure 6—figure supplement 2. Accumulation of 4MTB and I3M in X. laevis oocytes expressing close M. esculenta homologs of Me14G074000.Genes were expressed individually in X. laevis oocytes and transport activity was measured in the presence of 0.2 mM 4MTB (black bars) or 0.2 mM I3M (green bars). Accumulated 4MTB or I3M was quantified by LC-MS in 5 × 1 oocytes for each gene (Error bars represent ± s.d. of mean for data obtained from five different oocytes per experiment, experiment repeated two times). Dotted line represents media substrate concentration.DOI: http://dx.doi.org/10.7554/eLife.19466.023|
|Figure 6—figure supplement 3. Expression analysis of YFP-tagged (C-terminal) GTR homologs from cassava in X. laevis oocytes.YFP fluorescence in yellow (top) and the plasmamembrane-staining dye FM4-64fx in blue (bottom). Shown are representative images of at least three oocytes.DOI: http://dx.doi.org/10.7554/eLife.19466.024|
|Figure 7. Model of the evolution of the glucosinolate NPF transporter specificity.We propose that diversification of an ancestral high-affinity cyanogenic glucoside transporter (exemplified by MeCGTR1) lead to a dual-specificity transporter capable of transporting both cyanogenic glucosides and glucosinolates (exemplified by Me14G074000). With the emergence of glucosinolate biosynthesis, high-affinity, broad-specific glucosinolate transporters evolved (exemplified by CpGTRL1/2 and At/BrGTR1), which then further specialized to preferentially transport indole glucosinolates when indole biosynthesis emerged. Bidirectional arrow indicates an alternative model where high-affinity cyanogenic glucoside transporters emerged from the dual-specificity transporter (exemplified by Me14g074000). A. thaliana and C. papaya or M. esculenta diversified 108 MYA (median, 26 studies) or 72.1 MYA (median, 8 studies), respectively (Hedges et al., 2006). Branch points represent likely duplication events that led to new transporter substrate specificities. Striped pattern indicates a transporter that is unable to over-accumulate substrate compared to external media.DOI: http://dx.doi.org/10.7554/eLife.19466.025|
|Figure 8. Putative substrate binding site of GTR1, GTR3, Me14g74000 and MeCGTR1.Homology modeling of GTR1, GTR3, Me14g74000 and MecGTR1 was carried out using NPF6.3 as template (see Materials and methods for details). Residues P1–13 are shown and color-coded according to legend. In blue mesh is the 3V determined central cavity (Voss and Gerstein, 2010). The inserts show P4, P5 and P6 (see text for discussion).DOI: http://dx.doi.org/10.7554/eLife.19466.026Figure 8—figure supplement 1. Alignment of glucosinolate and cyanogenic glucoside transporters with NPF6.3 and selected POT transporters.Amino acid alignment of AtGTR1, AtGTR2, AtGTR3, CpGTRL1, CpGTRL2, Me14g74000 and MeCGTR1 with NPF6.3, PepTSo, PepTSt, PepT1 (human) and PepTGk using MUSCLE (Edgar, 2004) and visualized with JalView (Waterhouse et al., 2009). Residues involved in nitrate and peptide binding are highlighted by a star and numbered P1-P15 (Doki et al., 2013; Solcan et al., 2012; Parker and Newstead, 2014; Sun et al., 2014; Newstead, 2011; Lyons et al., 2014). Helixes are indicated above the alignment and based on NPF6.3 annotation.DOI: http://dx.doi.org/10.7554/eLife.19466.027|
|Figure 9. Root-mean square deviations (RMSD) of the position for all backbone atoms of the models 579 from their initial configuration as a function of simulation time.DOI: http://dx.doi.org/10.7554/eLife.19466.029|