Functional characterization of a Na+-dependent aspartate transporter from Pyrococcus horikoshii

Renae M Ryan, Emma L R Compton, Joseph A Mindell

Research output: Contribution to journalArticle

72 Citations (Scopus)

Abstract

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 GltPh, 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 GltPh 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 GltPh transport. We confirm that GltPh 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 GltPh 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 GltPh, and where an ion- and substrate-free transporter reorients to complete the transport cycle.

LanguageEnglish
Pages17540-17548
Number of pages9
JournalJournal of Biological Chemistry
Volume284
Issue number26
DOIs
Publication statusPublished - 26 Jun 2009

Fingerprint

Pyrococcus horikoshii
Amino Acid Transport Systems
Aspartic Acid
Excitatory Amino Acids
Ions
Amino Acid Transport System X-AG
Poisons
Substrates
Liposomes
Neurotransmitter Agents
Glutamic Acid
Proteins
Crystal structure
Amino Acids
Molecules
Kinetics

Keywords

  • pyrococcus horikoshii
  • amino acid transporters
  • glutamate

Cite this

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abstract = "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 GltPh, 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 GltPh 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 GltPh transport. We confirm that GltPh 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 GltPh 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 GltPh, and where an ion- and substrate-free transporter reorients to complete the transport cycle.",
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Functional characterization of a Na+-dependent aspartate transporter from Pyrococcus horikoshii. / Ryan, Renae M; Compton, Emma L R; Mindell, Joseph A.

In: Journal of Biological Chemistry, Vol. 284, No. 26, 26.06.2009, p. 17540-17548.

Research output: Contribution to journalArticle

TY - JOUR

T1 - Functional characterization of a Na+-dependent aspartate transporter from Pyrococcus horikoshii

AU - Ryan, Renae M

AU - Compton, Emma L R

AU - Mindell, Joseph A

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N2 - 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 GltPh, 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 GltPh 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 GltPh transport. We confirm that GltPh 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 GltPh 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 GltPh, and where an ion- and substrate-free transporter reorients to complete the transport cycle.

AB - 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 GltPh, 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 GltPh 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 GltPh transport. We confirm that GltPh 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 GltPh 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 GltPh, and where an ion- and substrate-free transporter reorients to complete the transport cycle.

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