| Summary: | Many proteins are localized at the vacuolar membrane, but most of them are still poorly described, due to the inaccessibility of this membrane from the extracellular environment. This work focused on the characterization of the CAT2 transporter from <i>S. lycopersicum</i> (<i>Sl</i>CAT2) that was previously overexpressed in <i>E. coli</i> and reconstituted in proteoliposomes for transport assay as [<sup>3</sup>H]Arg uptake. The orientation of the reconstituted transporter has been attempted and current data support the hypothesis that the protein is inserted in the liposome in the same orientation as in the vacuole. <i>Sl</i>CAT2 activity was dependent on the pH, with an optimum at pH 7.5. <i>Sl</i>CAT2 transport activity was stimulated by the increase of internal osmolality from 0 to 175 mOsmol while the activity was inhibited by the increase of external osmolality. K<sup>+</sup>, Na<sup>+</sup>, and Mg<sup>2+</sup> present on the external side of proteoliposomes at physiological concentrations, inhibited the transport activity; differently, the cations had no effect when included in the internal proteoliposome compartment. This data highlighted an asymmetric regulation of <i>Sl</i>CAT2. Cholesteryl hemisuccinate, included in the proteoliposomal membrane, stimulated the <i>Sl</i>CAT2 transport activity. The homology model of the protein was built using, as a template, the 3D structure of the amino acid transporter <i>Gk</i>ApcT. Putative substrate binding residues and cholesterol binding domains were proposed. Altogether, the described results open new perspectives for studying the response of <i>Sl</i>CAT2 and, in general, of plant vacuolar transporters to metabolic and environmental changes.
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