Analysis of the plastidic phosphate translocator gene family in Arabidopsis and identification of new phosphate translocator-homologous transporters, classified by their putative substrate-binding site

Abstract

Analysis of the Arabidopsis genome revealed the complete set of plastidic phosphate translocator (pPT) genes. The Arabidopsis genome contains 16 pPT genes: single copies of genes coding for the triose phosphate/phosphate translocator and the xylulose phosphate/phosphate translocator, and two genes coding for each the phosphoenolpyruvate/phosphate translocator and the glucose-6-phosphate/phosphate translocator. A relatively high number of truncated phosphoenolpyruvate/phosphate translocator genes (six) and glucose-6-phosphate/phosphate translocator genes (four) could be detected with almost conserved intron/exon structures as compared with the functional genes. In addition, a variety of PT-homologous (PTh) genes could be identified in Arabidopsis and other organisms. They all belong to the drug/metabolite transporter superfamily showing significant similarities to nucleotide sugar transporters (NSTs). The pPT, PTh, and NST proteins all possess six to eight transmembrane helices. According to the analysis of conserved motifs in these proteins, the PTh proteins can be divided into (a) the lysine (Lys)/arginine group comprising only non-plant proteins, (b) the Lys-valine/alanine/glycine group of Arabidopsis proteins, (c) the Lys/asparagine group of Arabidopsis proteins, and (d) the Lys/threonine group of plant and non-plant proteins. None of these proteins have been characterized so far. The analysis of the putative substrate-binding sites of the pPT, PTh, and NST proteins led to the suggestion that all these proteins share common substrate-binding sites on either side of the membrane each of which contain a conserved Lys residue.

Figure 1

Exon/intron structure of pPT genes and pseudogenes of Arabidopsis, rice, and potato. Alignment of deduced amino acid (aa) sequences of the genes depicting their structure within and between the pPT families. Homologous regions are indicated by bars (TPTs, black bars; PPTs, dark gray bars; GPTs, light gray bars; and XPT, white bar). Interrupted bars in the pseudogenes indicate lack of amino acid homology in comparison with the particular functional gene. The arrow marks the processing sites. Intron positions are indicated by triangles. Gray triangles indicate conserved intron positions. Accession numbers for genes from rice and potato are: OsTPT, OSJNBa0010K01; OsPPT, P0583G08; and StGPT, AY163867).

Figure 2

Expression of PT genes and pseudogenes. Specific primer combinations were used to amplify genomic DNA (G) and cDNA (C) from Arabidopsis. The numbers (500 and 1,000) indicate the length in base pairs. Differences in the length of G and C for one gene are due to intron sequences within the genomic DNA.

Figure 3

Phylogenetic tree of pPT, NST, and PTh families constructed by the neighbor-joining method. Amino acid sequences were aligned and the tree was created based on corrected distances using the programs ClustalX (v1.81) and TreeView v1.1.6. The numbers at the branches of the tree are bootstrap values (percentage; 1,000 repeats were performed). The first two letters of each sequence represent the organism: Ag, Anopheles gambiae; At, Arabidopsis; Dm, fruitfly (Drosophila melanogaster); Hs, human (Homo sapiens); Lm, Leishmania major; Ft, Flaveria trinervia; Gs, Galdieria sulfuraria; Nc, Neurospora crassa; Os, rice; Sc, Brewer's yeast (Saccharomyces cerevisiae); Sp, Fission yeast (Schizosaccharomyces pombe). At numbers are indicated except for the pPts from Arabidopsis (see Table I). Accession numbers not mentioned within the tree are the following: HsGDP-Fuc transporter (Q96A29), Dm-UDP sugar transporter (Q95YI5), Sc-sly41 (CAA38144), SpPT (CAB36873), OsTPT (Bac clone OSJNBa0010K01), OsPPT (AAK51561), and GsPT (phosphate translocator of unknown substrate specificity; A. Weber, unpublished data). Proteins from organisms other than Arabidopsis are marked in bold/italics. Already characterized proteins are marked by an asterisk.

Figure 4

Alignment of pPT, PTh, and NST amino acid sequences. The TPT sequence from spinach (Spinacia oleracea; SoTPT, accession no. CAA32016) was aligned with sequences of TPT from pea (Pisum sativum; PsTPT, accession no. CAA48210), PPT1 from Arabidopsis (AtPPT1, At5g33320) and tobacco (NtPPT, accession no. AAB40648), GPT from maize (Zea mays; ZmGPT, accession no. AF020813) and Arabidopsis (AtGPT, accession no. At5g54800), XPT from Arabidopsis (AtXPT, accession no. At5g17630), two proteins from fruitfly (DmCG14, accession no. AAF50956) and human (HSBAB55, accession no. BAB55306) representing the KR family, six proteins from Arabidopsis (At1g12500, At1g21870, At1g06890, At5g55950, At3g17430, and At1g53660) that are members of the KV/A/G, KT, and KD families, the human UDP-glucuronic acid transporter (HsK0260, accession no. BAB18586), the human GDP-Fuc transporter (HsGDPFu, accession no. Q96A29), and the GDP-Man transporter from Arabidopsis (AtGDPMa, accession no. At2g13650). Identities of amino acid residues are indicated by dots. The numbers refer to the amino acid position in the SoTPT sequence. The locations of seven transmembrane helices are indicated by solid lines (I-VII), whereas an eighth potential membrane-spanning region is indicated by a dashed line. Five regions of high similarities between the pPT proteins were boxed (white boxes). Lys and Arg residues that are conserved in all pPT proteins are marked by black boxes, whereas K41 and K273, which are probably involved in substrate binding, are marked by an arrow. Other residues that are conserved in most families of the TPT/NST superfamily are marked by gray boxes.

Figure 5

Localization of pPT genes and pseudogenes on the five chromosomes of Arabidopsis. A map of Arabidopsis chromosomes is shown. Centromeres are indicated by white boxes. pPT genes and clones containing pseudogenes are indicated by bars (gray bars, PPT gene family; striped bars, GPT gene family; and white bars, other members of the pPT gene family). Functional genes are underlined. Black lines connect PPT genes and pseudogenes of highest homology, whereas dotted lines connect the most closely related GPT genes and pseudogenes. Numbers at the lines represent the percentage of identity at amino acid levels.