Publications

• Large scale production of the active human ASCT2 (SLC1A5) transporter in Pichia pastoris--functional and kinetic asymmetry revealed in proteoliposomes.

  Pingitore P., Pochini L., Scalise M., Galluccio M., Hedfalk K., Indiveri C.

  The human glutamine/neutral amino acid transporter ASCT2 (hASCT2) was over-expressed in Pichia pastoris and purified by Ni(2+)-chelating and gel filtration chromatography. The purified protein was reconstituted in liposomes by detergent removal with a batch-wise procedure. Time dependent [(3)H]glu ... >> More

  The human glutamine/neutral amino acid transporter ASCT2 (hASCT2) was over-expressed in Pichia pastoris and purified by Ni(2+)-chelating and gel filtration chromatography. The purified protein was reconstituted in liposomes by detergent removal with a batch-wise procedure. Time dependent [(3)H]glutamine/glutamine antiport was measured in proteoliposomes which was active only in the presence of external Na(+). Internal Na(+) slightly stimulated the antiport. Optimal activity was found at pH7.0. A substantial inhibition of the transport was observed by Cys, Thr, Ser, Ala, Asn and Met (≥70%) and by mercurials and methanethiosulfonates (≥80%). Heterologous antiport of [(3)H]glutamine with other neutral amino acids was also studied. The transporter showed asymmetric specificity for amino acids: Ala, Cys, Val, Met were only inwardly transported, while Gln, Ser, Asn, and Thr were transported bi-directionally. From kinetic analysis of [(3)H]glutamine/glutamine antiport Km values of 0.097 and 1.8mM were measured on the external and internal sides of proteoliposomes, respectively. The Km for Na(+) on the external side was 32mM. The homology structural model of the hASCT2 protein was built using the GltPh of Pyrococcus horikoshii as template. Cys395 was the only Cys residue externally exposed, thus being the potential target of SH reagents inhibition and, hence, potentially involved in the transport mechanism. << Less

  Biochim. Biophys. Acta 1828:2238-2246(2013) [PubMed] [EuropePMC]

  This publication is cited by 7 other entries.

• The Human SLC1A5 Neutral Amino Acid Transporter Catalyzes a pH-Dependent Glutamate/Glutamine Antiport, as Well.

  Scalise M., Mazza T., Pappacoda G., Pochini L., Cosco J., Rovella F., Indiveri C.

  ASCT2 is a neutral amino acid transporter, which catalyzes a sodium-dependent obligatory antiport among glutamine and other neutral amino acids. The human ASCT2 over-expressed in <i>Pichia pastoris</i> and reconstituted in proteoliposomes has been employed for identifying alternative substrates of ... >> More

  ASCT2 is a neutral amino acid transporter, which catalyzes a sodium-dependent obligatory antiport among glutamine and other neutral amino acids. The human ASCT2 over-expressed in <i>Pichia pastoris</i> and reconstituted in proteoliposomes has been employed for identifying alternative substrates of the transporter. The experimental data highlighted that hASCT2 also catalyzes a sodium-dependent antiport of glutamate with glutamine. This unconventional antiport shows a preferred sidedness: glutamate is inwardly transported in exchange for glutamine transported in the counter direction. The orientation of the transport protein in proteoliposomes is the same as in the cell membrane; then, the observed sidedness corresponds to the transport of glutamate from the extracellular to the intracellular compartment. The competitive inhibition exerted by glutamate on the glutamine transport together with the docking analysis indicates that the glutamate binding site is the same as that of glutamine. The affinity for glutamate is lower than that for neutral amino acids, while the transport rate is comparable to that measured for the asparagine/glutamine antiport. Differently from the neutral amino acid antiport that is insensitive to pH, the glutamate/glutamine antiport is pH-dependent with optimal activity at acidic pH on the external (extracellular) side. The stimulation of glutamate transport by a pH gradient suggests the occurrence of a proton flux coupled to the glutamate transport. The proton transport has been detected by a spectrofluorometric method. The rate of proton transport correlates well with the rate of glutamate transport indicating a 1:1 stoichiometry H⁺: glutamate. The glutamate/glutamine antiport is also active in intact HeLa cells. On a physiological point of view, the described antiport could have relevance in some districts in which a glutamate/glutamine cycling is necessary, such as in placenta. << Less

  Front. Cell Dev. Biol. 8:603-603(2020) [PubMed] [EuropePMC]

  This publication is cited by 2 other entries.