title: Co-compartmentalization of the astroglial glutamate transporter, GLT-1, with glycolytic enzymes and mitochondria.
abstract: Efficient excitatory transmission depends on a family of transporters that use the Na(+)-electrochemical gradient to maintain low synaptic concentrations of glutamate. These transporters consume substantial energy in the spatially restricted space of fine astrocytic processes. GLT-1 (EAAT2) mediates the bulk of this activity in forebrain. To date, relatively few proteins have been identified that associate with GLT-1. In the present study, GLT-1 immunoaffinity isolates were prepared from rat cortex using three strategies and analyzed by liquid chromatography-coupled tandem mass spectrometry. In addition to known interacting proteins, the analysis identified glycolytic enzymes and outer mitochondrial proteins. Using double-label immunofluorescence, GLT-1 was shown to colocalize with the mitochondrial matrix protein, ubiquinol-cytochrome c reductase core protein 2 or the inner mitochondrial membrane protein, ADP/ATP translocase, in rat cortex. In biolistically transduced hippocampal slices, fluorescently tagged GLT-1 puncta overlapped with fluorescently tagged mitochondria along fine astrocytic processes. In a Monte Carlo-type computer simulation, this overlap was significantly more frequent than would occur by chance. Furthermore, fluorescently tagged hexokinase-1 overlapped with mitochondria or GLT-1, strongly suggesting that GLT-1, mitochondria, and the first step in glycolysis are cocompartmentalized in astrocytic processes. Acute inhibition of glycolysis or oxidative phosphorylation had no effect on glutamate uptake in hippocampal slices, but simultaneous inhibition of both processes significantly reduced transport. Together with previous results, these studies show that GLT-1 cocompartmentalizes with Na(+)/K(+) ATPase, glycolytic enzymes, and mitochondria, providing a mechanism to spatially match energy and buffering capacity to the demands imposed by transport.
authors.affiliation: Children's Hospital of Philadelphia Research Institute, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA.
authors.forename: Elizabeth N
authors.initials: EN
authors.lastname: Genda
authors.forename: Joshua G
authors.initials: JG
authors.lastname: Jackson
authors.forename: Amanda L
authors.initials: AL
authors.lastname: Sheldon
authors.forename: Susannah F
authors.initials: SF
authors.lastname: Locke
authors.forename: Todd M
authors.initials: TM
authors.lastname: Greco
authors.forename: John C
authors.initials: JC
authors.lastname: O'Donnell
authors.forename: Lynn A
authors.initials: LA
authors.lastname: Spruce
authors.forename: Rui
authors.initials: R
authors.lastname: Xiao
authors.forename: Wensheng
authors.initials: W
authors.lastname: Guo
authors.forename: Mary
authors.initials: M
authors.lastname: Putt
authors.forename: Steven
authors.initials: S
authors.lastname: Seeholzer
authors.forename: Harry
authors.initials: H
authors.lastname: Ischiropoulos
authors.forename: Michael B
authors.initials: MB
authors.lastname: Robinson
mesh_terms: D000818:Animals; D001253:Astrocytes / Q000378:metabolism*; D002478:Cells, Cultured; D002540:Cerebral Cortex / Q000378:metabolism; D014450:Electron Transport Complex III / Q000378:metabolism; D027342:Excitatory Amino Acid Transporter 2 / Q000378:metabolism*; D018698:Glutamic Acid / Q000378:metabolism; D006593:Hexokinase / Q000378:metabolism*; D006624:Hippocampus / Q000378:metabolism; D008297:Male; D008928:Mitochondria / Q000378:metabolism*; D000226:Mitochondrial ADP, ATP Translocases / Q000378:metabolism; D009474:Neurons / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley
chemical_list: D027342:Excitatory Amino Acid Transporter 2; D018698:Glutamic Acid; D000226:Mitochondrial ADP, ATP Translocases; D006593:Hexokinase; D014450:Electron Transport Complex III