Functional Characterization of a Na+-dependent Aspartate Transporter from Pyrococcus horikoshii

Excitatory amino acid transporters (EAATs) are crucial in maintaining extracellular levels of glutamate, the most abundant excitatory neurotransmitter, below toxic levels. The recent three-dimensional crystal structure of Glt(Ph), an archaeal homolog of the EAATs, provides elegant structural details of this family of proteins, yet we know little about the mechanism of the bacterial transporter. Conflicting reports in the literature have described Glt(Ph) as an aspartate transporter driven by Na+ or a glutamate transporter driven by either Na+ or H+. Here we use purified protein reconstituted into liposomes to thoroughly characterize the ion and substrate dependence of the Glt(Ph) transport. We confirm that Glt(Ph) is a Na+-dependent transporter that is highly selective for aspartate over other amino acids, and we show that transport is coupled to at least two Na+ ions. In contrast to the EAATs, transport via Glt(Ph) is independent of H+ and K+. We propose a kinetic model of transport in which at least two Na+ ions are coupled to the cotransport of each aspartate molecule by Glt(Ph), and where an ion-and substrate-free transporter reorients to complete the transport cycle.