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Heterologously Expressed GLT-1 Associates in 200-nm Protein-Lipid Islands

Stefan Raunser ^*, Winfried Haase ^*, Cornelia Franke , Gunter P. Eckert , Walter E. Müller  and Werner Kühlbrandt ^*

^* Department of Structural Biology, Max-Planck-Institute of Biophysics, Frankfurt am Main, Germany; and Department of Pharmacology, Biocenter Niederursel, University of Frankfurt, Frankfurt am Main, Germany

Correspondence: Address reprint requests to Dr. Werner Kühlbrandt, Dept. of Structural Biology, Max-Planck-Institute of Biophysics, Max-von-Laue-Str. 3, 60438 Frankfurt am Main, Germany. E-mail: werner.kuehlbrandt{at}mpibp-frankfurt.mpg.de.

The glutamate transporter GLT-1 from Rattus norvegicus was expressed at high level in baby hamster kidney (BHK-21) cells by the Semliki Forest Virus expression system. We examined the expressed GLT-1 in the plasma membrane and found that the transporter accumulates in detergent-insoluble lipid-protein assemblies. Freeze-fracture, immunogold labeling, and electron microscopy revealed that GLT-1 forms 200-nm protein-rich islands in the plasma membrane. Cholesterol depletion in living cells resulted in a dispersion of the GLT-1 islands, indicating that they are the result of lipid-protein rather than protein-protein interactions. Disruption of GLT-1 islands and dispersion of GLT-1 goes along with a reduction of the glutamate transport activity. Our direct visualization of lipid-protein islands in the plasma membrane of tissue culture cells suggests that the reported clustering of glutamate transporters and their cholesterol-dependent transport activity in cells is likewise connected to their association with cholesterol-rich microdomains in the plasma membrane.