Regulatory Aspects of the Vacuolar CAT2 Arginine Transporter of S. lycopersicum: Role of Osmotic Pressure and Cations.

Jessica Cosco,Michele Galluccio,Teresa M R Regina,Cesare Indiveri,Mariafrancesca Scalise

doi:10.3390/ijms20040906

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 S. lycopersicum (SlCAT2) that was previously overexpressed in E. coli 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. SlCAT2 activity was dependent on the pH, with an optimum at pH 7.5. SlCAT2 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 Mg2+ 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 SlCAT2. Cholesteryl hemisuccinate, included in the proteoliposomal membrane, stimulated the SlCAT2 transport activity. The homology model of the protein was built using, as a template, the 3D structure of the amino acid transporter GkApcT. Putative substrate binding residues and cholesterol binding domains were proposed. Altogether, the described results open new perspectives for studying the response of SlCAT2 and, in general, of plant vacuolar transporters to metabolic and environmental changes.

Highlights

The vacuole is the largest organelle in plant cells and it plays several roles
To assess the orientation of the SlCAT2 transporter in the proteoliposomal membrane, a method based on side-specific targeting was employed [33]
Transport inhibition was observed upon incubation of the antibody with the protein before the insertion in the membrane, while no significant effect could be observed upon addition of the antibody to the external side (Figure 1A)

Summary

Introduction

The vacuole is the largest organelle in plant cells and it plays several roles. Originally, this organelle was considered mainly responsible for cell turgor; it became clear that the vacuole is involved in protein digestions, storage of water, ions, and metabolites as well as toxic compounds [1,2]. The vacuole received more attention because is involved in plant metabolism, pH homeostasis, stress responses, cell growth and development [2,3,4], and signal transduction [3,5]. In this frame, many proteins are expected to accomplish these functions [1,3]. The characterization of transporters in the vacuolar membrane is not straightforward due to the inaccessibility of these transporters from the extracellular environment. The vacuolar transporter CAT2 from S. lycopersicum (SlCAT2) has been recently characterized in the in vitro system of proteoliposomes obtained by reconstituting the recombinant protein over-expressed in E. coli [7]

Methods

Results

Conclusion