PMID:30791488

Citation	Cosco, J, Regina, TMR, Scalise, M, Galluccio, M and Indiveri, C (2019) Regulatory Aspects of the Vacuolar CAT2 Arginine Transporter of : Role of Osmotic Pressure and Cations. Int J Mol Sci 20
Abstract	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 (CAT2) that was previously overexpressed in and reconstituted in proteoliposomes for transport assay as [³H]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. CAT2 activity was dependent on the pH, with an optimum at pH 7.5. 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⁺, Na⁺, and Mg 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 CAT2. Cholesteryl hemisuccinate, included in the proteoliposomal membrane, stimulated the CAT2 transport activity. The homology model of the protein was built using, as a template, the 3D structure of the amino acid transporter ApcT. Putative substrate binding residues and cholesterol binding domains were proposed. Altogether, the described results open new perspectives for studying the response of CAT2 and, in general, of plant vacuolar transporters to metabolic and environmental changes.
Links	PubMed PMC6413183 Online version:10.3390/ijms20040906
Keywords	Arginine/metabolism; Biological Transport; Cationic Amino Acid Transporter 2/administration & dosage; Cationic Amino Acid Transporter 2/chemistry; Cationic Amino Acid Transporter 2/genetics; Cationic Amino Acid Transporter 2/metabolism; Cations/metabolism; Dose-Response Relationship, Drug; Escherichia coli/genetics; Escherichia coli/metabolism; Hydrogen-Ion Concentration; Liposomes; Models, Molecular; Molecular Conformation; Osmotic Pressure; Recombinant Proteins/administration & dosage; Recombinant Proteins/genetics; Recombinant Proteins/metabolism; Structure-Activity Relationship; Vacuoles/metabolism
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Annotations

Gene product	Qualifier	GO Term	Evidence Code	with/from	Aspect	Extension	Notes	Status
SOLLC:I6UDU5		GO:0015171: amino acid transmembrane transporter activity	ECO:0005551: biological system reconstruction evidence by experimental evidence		F		Figure 1. Sidedness of the reconstituted Solanum lycopersicum CAT2 (SlCAT2) in proteoliposomes. (A) SlCAT2 was purified and reconstituted in proteoliposomes as described in Section 4.4.	complete

References

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