Heterologously expressed GLT-1 associates in ~200 nm protein-lipid islands
Stefan Raunser 1, Winfried Haase 1, Cornelia Franke 2, Gunter P Eckert 2, Walter E Müller 2 and Werner Kühlbrandt 1*
1 Max Planck Institute of Biophysics, Frankfurt, Germany
2 Department of Pharmacology, Biocenter Niederursel, University of Frankfurt
* To whom correspondence should be addressed. E-mail: werner.kuehlbrandt{at}mpibp-frankfurt.mpg.de.
Submitted on April 12, 2006
Revised on June 19, 2006
Accepted on 14 August 2006
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Abstract |
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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 labelling 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.
Key Words:
cholesterol, electron microscopy, glutamate transporter, lipid rafts, membrane protein
