Efficient excitatory transmission depends on a family of transporters that utilize 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 LC 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 co-localize with the mitochondrial matrix protein, ubiquinol-cytochrome c reductase core protein II (UQCRC2) or the inner mitochondrial membrane protein, ADP/ATP translocase (ANT), 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 co-compartmentalized 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 co-compartmentalizes with Na+/K+ ATPase, glycolytic enzymes, and mitochondria, providing a mechanism to spatially match energy and buffering capacity to the demands imposed by transport.

GLAST is the predominant glutamate transporter in the cerebellum and contributes substantially to glutamate transport in forebrain. This astroglial glutamate transporter quickly binds and clears synaptically released glutamate and is principally responsible for ensuring that synaptic glutamate concentrations remain low. This process is associated with a significant energetic cost. Compartmentalization of GLAST with mitochondria and proteins involved in energy metabolism could provide energetic support for glutamate transport. Therefore, we performed immunoprecipitation and co-localization experiments to determine if GLAST might co-compartmentalize with proteins involved in energy metabolism. GLAST was immunoprecipitated from rat cerebellum and subunits of the Na+/K+ ATPase, glycolytic enzymes, and mitochondrial proteins were detected. GLAST co-localized with mitochondria in cerebellar tissue. GLAST also co-localized with mitochondria in fine processes of astrocytes in organotypic hippocampal slice cultures. From these data, we hypothesized that mitochondria participate in a macromolecular complex with GLAST to support oxidative metabolism of transported glutamate. To determine the functional metabolic role of this complex, we measured CO2 production from radiolabeled glutamate in cultured astrocytes and compared it to overall glutamate uptake. Within 15 minutes, 9% of transported glutamate was converted to CO2. This CO2 production was blocked by inhibitors of glutamate transport and glutamate dehydrogenase, but not by an inhibitor of glutamine synthetase. Our data support a model in which GLAST exists in a macromolecular complex that allows transported glutamate to be metabolized in mitochondria to support energy production.