title: Transport mechanism and regulatory properties of the human amino acid transporter ASCT2 (SLC1A5).
abstract: The kinetic mechanism of the transport catalyzed by the human glutamine/neutral amino acid transporter hASCT2 over-expressed in P. pastoris was determined in proteoliposomes by pseudo-bi-substrate kinetic analysis of the Na(+)-glutamineex/glutaminein transport reaction. A random simultaneous mechanism resulted from the experimental analysis. Purified functional hASCT2 was chemically cross-linked to a stable dimeric form. The oligomeric structure correlated well with the kinetic mechanism of transport. Half-saturation constants (Km) of the transporter for the other substrates Ala, Ser, Asn and Thr were measured both on the external and internal side. External Km were much lower than the internal ones confirming the asymmetry of the transporter. The electric nature of the transport reaction was determined imposing a negative inside membrane potential generated by K(+) gradients in the presence of valinomycin. The transport reaction resulted to be electrogenic and the electrogenicity originated from external Na(+). Internal Na(+) exerted a stimulatory effect on the transport activity which could be explained by a regulatory, not a counter-transport, effect. Native and deglycosylated hASCT2 extracted from HeLa showed the same transport features demonstrating that the glycosyl moiety has no role in transport function. Both in vitro and in vivo interactions of hASCT2 with the scaffold protein PDZK1 were revealed.
authors: Department DiBEST (Biologia, Ecologia, Scienze della Terra) Unit of Biochemistry and Molecular Biotechnology, University of Calabria, Via P. Bucci 4C, 87036, Arcavacata di Rende, CS, Italy.
authors: Mariafrancesca
authors: M
authors: Scalise
authors: Lorena
authors: L
authors: Pochini
authors: Simona
authors: S
authors: Panni
authors: Piero
authors: P
authors: Pingitore
authors: Kristina
authors: K
authors: Hedfalk
authors: Cesare
authors: C
authors: Indiveri
mesh_terms: D026962:Amino Acid Transport System ASC / Q000378:metabolism*; D000596:Amino Acids / Q000737:chemistry*; D000818:Animals; D001692:Biological Transport; D002352:Carrier Proteins / Q000378:metabolism; D003432:Cross-Linking Reagents / Q000737:chemistry; D004563:Electrochemistry; D005786:Gene Expression Regulation; D005973:Glutamine / Q000737:chemistry; D006367:HeLa Cells; D006801:Humans; D007700:Kinetics; D008081:Liposomes / Q000737:chemistry; D008565:Membrane Proteins; D015778:Minor Histocompatibility Antigens; D010843:Pichia / Q000378:metabolism; D011188:Potassium / Q000737:chemistry; D051381:Rats; D011994:Recombinant Proteins / Q000378:metabolism; D012964:Sodium / Q000737:chemistry; D014634:Valinomycin / Q000737:chemistry
chemical_list: D026962:Amino Acid Transport System ASC; D000596:Amino Acids; D002352:Carrier Proteins; D003432:Cross-Linking Reagents; D008081:Liposomes; D008565:Membrane Proteins; D015778:Minor Histocompatibility Antigens; C484659:PDZK1 protein, human; D011994:Recombinant Proteins; C420506:SLC1A5 protein, human; D005973:Glutamine; D014634:Valinomycin; D012964:Sodium; D011188:Potassium