The Poly-Glutamate Motif of GmMATE4 Regulates Its Isoflavone Transport Activity

Abstract

Multidrug and toxic compound extrusion (MATE) transporters in eukaryotes have been characterized to be antiporters that mediate the transport of substrates in exchange for protons. In plants, alkaloids, phytohormones, ion chelators, and flavonoids have been reported to be the substrates of MATE transporters. Structural analyses have been conducted to dissect the functional significance of various motifs of MATE proteins. However, an understanding of the functions of the N- and C-termini has been inadequate. Here, by performing phylogenetic analyses and protein sequence alignment of 14 representative plant species, we identified a distinctive N-terminal poly-glutamate motif among a cluster of MATE proteins in soybean. Amongst them, GmMATE4 has the most consecutive glutamate residues at the N-terminus. A subcellular localization study showed that GmMATE4 was localized at the vacuolar membrane-like structure. Protein charge prediction showed that the mutation of the glutamate residues to alanine would reduce the negative charge at the N-terminus. Using yeast as the model, we showed that GmMATE4 mediated the transport of daidzein, genistein, glycitein, and glycitin. In addition, the glutamate-to-alanine mutation reduced the isoflavone transport capacity of GmMATE4. Altogether, we demonstrated GmMATE4 as an isoflavone transporter and the functional significance of the N-terminal poly-glutamate motif of GmMATE4 for regulating the isoflavone transport activity.

Keywords: acidic amino acid; isoflavone; legume; multidrug and toxic compound extrusion (MATE) transporter; poly-glutamate motif; proton gradient; proton motive force; soybean.

Figure 1

Phylogenetic analysis of the MATE family of proteins from 14 Leguminosae species (Arachis ipaensis, Cajanus cajan, Cicer arietinum, Lotus japonicus, Medicago truncatula, Phaseolus vulgaris, Vigna radiata, and Glycine max) and non-Leguminosae plants (Arabidopsis thaliana, Gossypium hirsutum, Helianthus annuus, Oryza sativa, Solanum lycopersicum, and Solanum tuberosum). The cluster consisting solely of MATE proteins from Glycine max is indicated by a black bar in clade 2. (A) The whole phylogenetic tree; (B) the enlarged portion of the phylogenetic tree showing clade 1; (C) the enlarged portion of the phylogenetic tree showing clade 2; (D) the enlarged portion of the phylogenetic tress showing clade 3; (E) the enlarged portion of the phylogenetic tree showing clade 4.

Figure 2

Alignment of the N-terminal amino acid sequences of Glyma.03G005200, Glyma.03G005300, Glyma.03G005400, GmMATE4, GmMATE1, and GmMATE2. The alignment of the full protein sequences is shown in Figure S2. The sequence alignment was done by ClustalW using MEGA11 [20].

Figure 3

Subcellular localization study of GmMATE4, GmMATE4Δ3ala, and GmMATE4Δ7ala. GFP was fused to the C-termini of GmMATE4, GmMATE4Δ3ala, and GmMATE4Δ7ala. The fusion construct was cloned downstream of a CaMV 35S promoter in the plasmid V7. The plasmid was coated onto gold particles, bombarded into onion epidermal cells, and then, observed using a confocal microscope. Scale bar, 50 μm; excitation 488 nm; and the emission signal was collected between 500 and 545 nm. All cells having the green, fluorescent signal (≥11 cells) from two biological repeats showed the same patterns.

Figure 4

Uptake assays of isoflavones. The isoflavones detected in the yeast cells after the treatments of: (A) daidzein; (B) genistein; (C) glycitein; or (D) glycitin. Error bar: standard error from three technical repeats. Student’s t test (two-tailed) was performed to compare the isoflavone uptake capacity between the constructs. **** indicates p < 0.0001. The experiments were performed twice with similar results.

Figure 5

Uptake assays of isoflavones. The isoflavones detected in the yeast cells after the treatments of (A) daidzein, (B) genistein, (C) glycitein, and (D) glycitin. Error bar: standard error from three technical repeats. Student’s t test (two-tailed) was performed to compare the isoflavone uptake capacity between the constructs. **** indicates p < 0.0001. The experiments were performed twice with similar results.