A chimera carrying the functional domain of the orphan protein SLC7A14 in the backbone of SLC7A2 mediates trans-stimulated arginine transport.
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.
Department of Pharmacology, University Medical Center of the Johannes Gutenberg University, 55131 Mainz, Germany.
Isabel
I
Jaenecke
Jean-Paul
JP
Boissel
Matthias
M
Lemke
Johanna
J
Rupp
Bruno
B
Gasnier
Ellen I
EI
Closs
D026923:Amino Acid Transport Systems, Basic / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D001120:Arginine / Q000235:genetics / Q000378:metabolism*; D001693:Biological Transport, Active / Q000502:physiology; D002460:Cell Line; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008247:Lysosomes / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012867:Skin / Q000166:cytology / Q000378:metabolism*; D014982:Xenopus laevis
D026923:Amino Acid Transport Systems, Basic; D011993:Recombinant Fusion Proteins; C414177:SLC7A2 protein, human; D001120:Arginine