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.2012 Aug 31;287(36):30853-60.
doi: 10.1074/jbc.M112.350322. Epub 2012 Jul 11.

A chimera carrying the functional domain of the orphan protein SLC7A14 in the backbone of SLC7A2 mediates trans-stimulated arginine transport

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A chimera carrying the functional domain of the orphan protein SLC7A14 in the backbone of SLC7A2 mediates trans-stimulated arginine transport

Isabel Jaenecke et al. J Biol Chem..

Abstract

In human skin fibroblasts, a lysosomal transport system specific for cationic amino acids has been described and named system c. We asked if SLC7A14 (solute carrier family 7 member A14), an orphan protein assigned to the SLC7 subfamily of cationic amino acid transporters (CATs) due to sequence homology, may represent system c. Fusion proteins between SLC7A14 and enhanced GFP localized to intracellular vesicles, co-staining with the lysosomal marker LysoTracker(®). To perform transport studies, we first tried to redirect SLC7A14 to the plasma membrane (by mutating putative lysosomal targeting motifs) but without success. We then created a chimera carrying the backbone of human (h) CAT-2 and the protein domain of SLC7A14 corresponding to the so-called "functional domain" of the hCAT proteins, a protein stretch of 81 amino acids that determines the apparent substrate affinity, sensitivity to trans-stimulation, and (as revealed in this study) pH dependence. The chimera mediated arginine transport and exhibited characteristics similar but not identical to hCAT-2A (the low affinity hCAT-2 isoform). Western blot and microscopic analyses confirmed localization of the chimera in the plasma membrane of Xenopus laevis oocytes. Noticeably, arginine transport by the hCAT-2/SLC7A14 chimera was pH-dependent, trans-stimulated, and inhibited by α-trimethyl-L-lysine, properties assigned to lysosomal transport system c in human skin fibroblasts. Expression analysis showed strong expression of SLC7A14 mRNA in these cells. Taken together, these data strongly suggest that SLC7A14 is a lysosomal transporter for cationic amino acids.

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Figures

FIGURE 1.
FIGURE 1.
Localization of SLC7A14 in human U373MG glioblastoma cells.A, U373MG cells overexpressing the SLC7A14-EGFP fusion protein (stable transfection;green) were stained with LysoTracker®, MitoTracker®, or ER-TrackerTM (red). Cells were analyzed by confocal laser scanning microscopy.Scale bars = 10 μm.B, U373MG cells overexpressing SLC7A14-EGFP were lysed and analyzed by Western blotting. Proteins with (+) or without (−) glycosidase treatment were separated by 7.5% SDS-PAGE, blotted, and probed with anti-GFP antibody (upper panel) and anti-tubulin antibody as a loading control (lower panel).
FIGURE 2.
FIGURE 2.
mRNA expression of SLC7A14 and hCATs in human skin fibroblasts. Total RNA was isolated from skin fibroblasts of six donors. mRNAs of SLC7 family members were measured by quantitative RT-PCR. GAPDH was chosen as the housekeeping gene for relative determinations and was set at 1.Bars represent means ± S.E. (n = 6).
FIGURE 3.
FIGURE 3.
Localization of SLC7A14, hCAT-2/A14_BK, and hCAT-2A inXenopus oocytes.A, scheme of the chimeric protein hCAT-2/A14_BK.B,Xenopus oocytes were frozen in tissue freezing medium 3 days after cRNA injection. Cryosections (12 μm) were analyzed by fluorescence microscopy.C, proteins ofXenopus oocytes expressing the EGFP fusion protein or EGFP alone as indicated were biotinylated at the cell surface. Non-injected oocytes served as controls. Subsequently, oocytes were lysed for Western blot analysis. Total lysates (left panels) and surface proteins (separated from non-biotinylated intracellular proteins by avidin-coated beads;right panels) were separated by 10% SDS-PAGE, blotted, and probed with anti-GFP antibody (upper panels) and subsequently with anti-tubulin antibody as a loading control (lower left panel) and successful withdrawal of intracellular proteins (lower right panel).
FIGURE 4.
FIGURE 4.
Trans-stimulation of hCAT-2/A14_BK in comparison with hCAT-1 and hCAT-2A.Xenopus oocytes expressing the indicated transporters were incubated in amino acid-free medium for 4–6 h and then injected with 36 nl of 100 mml-[3H]arginine (100 μCi/ml). Three oocytes each were incubated for 30 min in pH 7.5 buffer containing either no amino acids or 1 mm arginine. Subsequently, the radioactivity in the supernatants was determined. Values obtained with non-injected oocytes were subtracted, and the radioactivity in buffer containing no amino acids is expressed as a percentage of the respective radioactivity detected in the extracellular buffer containing 1 mml-arginine (100%,dashed line).Bars represent means ± S.E. (n = 9–15), with at least three different batches of oocytes. Statistical analysis was performed using analysis of variance with the Bonferroni post hoc test. ***,p ≤ 0.001;n.s., not significant.
FIGURE 5.
FIGURE 5.
pH dependence ofl-arginine transport mediated by hCAT-2/A14_BK in comparison with hCAT-1 and hCAT-2A.Xenopus oocytes were injected with cRNAs encoding individual transporters.A) the dependence of uptake of 1 mml-[3H]arginine into oocytes expressing hCAT-2/A14_BK (■), hCAT-2A (▴), hCAT-1 (○), or hCAT-2/1_BK (♢) on pH in the extracellular buffer was measured.B, 36 nl of 100 mml-[3H]arginine (100 μCi/ml) were injected into the oocytes. Efflux measured in the extracellular buffer containing 1 mm arginine at pH 5 is expressed as a percentage of efflux at pH 7.5 (dashed line).Data points andbars represent means ± S.E. (n = 10–20 for influx and 9–15 for efflux experiments), with at least two different batches of oocytes. Statistical analysis was performed using analysis of variance with the Bonferroni post hoc test. ***,p ≤ 0.001; **,p ≤ 0.01;n.s., not significant.
FIGURE 6.
FIGURE 6.
Transport activity in the presence of the system c inhibitor ϵ-trimethyl-l-lysine.Xenopus oocytes were injected with cRNAs encoding the indicated transporters. The uptake of 30 μml-[3H]arginine in the absence or presence of 3 mm ϵ-trimethyl-l-lysine was determined. Values obtained in the absence of the putative inhibitor were set as 100% (data not shown).Bars represent means ± S.E. (n = 10–20), with at least two different batches of oocytes. Statistical analysis was performed using analysis of variance with the Bonferroni post hoc test. ***,p ≤ 0.001; **,p ≤ 0.01 compared with untreated controls.
FIGURE 7.
FIGURE 7.
Substrate recognition of several chimeric proteins in comparison with the wild-type transporters.Xenopus oocytes were injected with cRNAs encoding the indicated transporters.A, the uptake of 1 mml-[3H]arginine (black bars) or 1 mm [3H]histidine (gray bars) was measured at pH 5.5.Data points represent means ± S.E. (n = 13–28), with at least two different batches of oocytes.B, the same data as shown inA, but histidine transport is expressed as a percentage of the arginine transport measured in parallel by the same transporter.
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References

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