Characterization of the preprotein and amino acid transporter gene family in Arabidopsis

Abstract

Seventeen loci encode proteins of the preprotein and amino acid transporter family in Arabidopsis (Arabidopsis thaliana). Some of these genes have arisen from recent duplications and are not in annotated duplicated regions of the Arabidopsis genome. In comparison to a number of other eukaryotic organisms, this family of proteins has greatly expanded in plants, with 24 loci in rice (Oryza sativa). Most of the Arabidopsis and rice genes are orthologous, indicating expansion of this family before monocot and dicot divergence. In vitro protein uptake assays, in vivo green fluorescent protein tagging, and immunological analyses of selected proteins determined either mitochondrial or plastidic localization for 10 and six proteins, respectively. The protein encoded by At5g24650 is targeted to both mitochondria and chloroplasts and, to our knowledge, is the first membrane protein reported to be targeted to mitochondria and chloroplasts. Three genes encoded translocase of the inner mitochondrial membrane (TIM)17-like proteins, three TIM23-like proteins, and three outer envelope protein16-like proteins in Arabidopsis. The identity of Arabidopsis TIM22-like proteins is most likely a protein encoded by At3g10110/At1g18320, based on phylogenetic analysis, subcellular localization, and complementation of a yeast (Saccharomyces cerevisiae) mutant and coexpression analysis. The lack of a preprotein and amino acid transporter domain in some proteins, localization in mitochondria, plastids, or both, variation in gene structure, and the differences in expression profiles indicate that the function of this family has diverged in plants beyond roles in protein translocation.

Figure 1.

Multiple sequence alignment of the predicted proteins of the PRAT family. Proteins were aligned using ClustalX with gaps introduced to maximize alignment and the TIM17, 22, and 23 proteins from yeast and OEP16 from pea included as a comparison. Predicted transmembrane regions are highlighted in gray; the PRAT domain is indicated above the sequence where X indicates any amino acid. The positions used for phylogenetic analysis are marked with asterisks below the sequences. Residues are colored according to properties: red, hydrophobic (AVFPMILW); blue, acidic (DE); magenta, basic (RHK); green, hydrophilic (STYCNGQ).

Figure 2.

Phylogenetic analysis and gene structure of genes encoding PRAT proteins. A, Neighbor-joining tree of the Arabidopsis PRAT protein family with yeast TIM17, 22, 23, and pea OEP16 included for comparison. The tree was constructed using 82 amino acid positions in the region of transmembrane helices 2 to 4 as marked in Figure 1. Only bootstrap values above 50% are shown. B, Gene structure of the various genes belonging to the TIM17, 22, and 23 family of proteins in TAIR 6. [See online article for color version of this figure.]

Figure 3.

In vitro and in vivo subcellular localizations of Arabidopsis TIM17 and TIM23 proteins. The ability to import proteins in vitro was assessed by incubating radiolabeled precursor proteins with mitochondria or chloroplasts under conditions that support protein uptake into each organelle. The uptake of rbcS was used as a positive control for chloroplast import and the uptake of the phosphate translocator from maize (ZmPic) was used as a positive control for mitochondrial import. Attaching GFP in frame to the C-terminal end of the protein and transformation of suspension cells by biolistic bombardment and visualization by fluoresence microscopy assessed in vivo targeting ability. The targeting signals of Aox and rbcS attached to the RFP (Aox-RFP and rbcS-RFP) were used as controls for mitochondria and plastids, respectively. For mitochondrial import, in vitro import into mitochondria was followed by rupture of the outer membrane to verify insertion into the inner membrane. Addition of compounds to the import assay are indicated; the presence of Mit and Mit*OM (lanes 6–9; boxed) indicates that mitochondria were ruptured after the import assay, but prior to protease treatment. For chloroplast import, precursors were incubated with purified chloroplast followed by thermolysin treatment as indicated. Unless otherwise indicated, RFP (middle) is the pattern obtained with Aox-RFP. Mit, Mitochondria; Mit*OM, outer membrane-ruptured mitochondria; Val, valinomycin; PK, proteinase K; Chloro, chloroplasts; Therm, thermolysin. Sizes are indicated as apparent molecular mass in kilodaltons.

Figure 4.

In vitro and in vivo subcellular localizations of unknown Arabidopsis PRAT proteins. N terminus indicates that GFP was fused in frame to the N-terminal end of the protein. The adenine nucleotide translocator from maize was used as a control for mitochondrial import.

Figure 5.

Immunological analysis of mitochondria and chloroplasts with antibodies raised to proteins encoded by At3g49560 and At4g26670. Immunological analysis of PRAT proteins encoded by At4g26670, At5g55510, At3g49560, At5g24650, At2g28900, At4g16160, At2g42210, and At3g25120 (lanes 1–8). A, Coomassie-stained gel (1 μg protein/lane). B, Western blot with antibodies raised against the protein encoded by At4g26670 (0.1 μg protein/lane). C, Western blot with antibodies raised against the protein encoded by At3g49560 (0.1 μg protein/lane). Lanes 1 to 6, Full-length proteins fused with a C-terminal His tag expressed in Escherichia coli and purified using a nickel-nitrilotriacetic acid column. Lanes 7 to 8, Crude E. coli lysates containing partial proteins fused with an N-terminal glutathione S-transferase tag. D, Mitochondrial and chloroplastidic proteins were separated by SDS-PAGE and probed with various antibodies. VDAC, Mitochondrial voltage-dependent anion channel; UCP, mitochondrial inner membrane uncoupling protein.

Figure 6.

Complementation of yeast lacking a functional TIM22 protein. The ability of mitochondrially targeted PRAT proteins from Arabidopsis to complement yeast lacking TIM22 was tested using a replacement strategy. Expression of the yeast TIM22 was dependent on the presence of Gal in the medium. The ability of the Arabidopsis genes to complement was tested by expressing them under a constitutive promoter in yeast and growing in a medium lacking Gal. The ability to complement was evidenced by growth. The yeast gene (ScTIM22) acts as a positive control and the empty vector acts as a negative control.

Figure 7.

Self-organizing map analysis of the expression patterns of genes encoding mitochondria (red), chloroplast (green), dual-targeted (red and green), cytosolic (brown), peroxisomal (blue), and nuclear (magenta) proteins. QRT-PCR analysis was carried out on RNA isolated from second rosette leaves at seven stages of development: just emerged 0, 1, 2, 3, 4, 5, and 6 weeks old. The amount of mRNA for each transcript was normalized where the maximal value was set to 100 and other values were expressed relative to it. The blue line represents the pattern of transcript abundance with variation indicated by red lines. Members of the PRAT family are indicated in black with a colored square used to indicate the subcellular location as defined in Figures 3 to 5. Because At1g18320 and At3g10110 display 100% nucleic acid sequence homology, it was not possible to develop a QRT-PCR assay that was gene specific and thus they were omitted from this analysis. Transcripts of At4g16160 could not be detected because this gene is not expressed in rosette leaves. Gene abbreviations are listed in Supplemental Table S1.