High-yield Expression, Reconstitution and Structure of the Recombinant, Fully Functional Glutamate Transporter GLT-1 from Rattus norvegicus Stefan Raunser, Winfried Haase, Mihnea Bostina, David N. Parcej and Werner Ku ¨ hlbrandt * Department of Structural Biology, Max-Planck-Institute of Biophysics, 60439 Frankfurt am Main, Germany The glutamate transporter GLT-1 from Rattus norvegicus was expressed at high level in BHK cells using the Semliki Forest virus expression system. BHK cells infected with viral particles carrying the GLT-1 gene exhibited 30-fold increased aspartate uptake compared to control cells. The expression level of GLT-1 as determined by binding of labelled substrate to membrane preparations was about 3.5!10 6 functional transporters per cell, or 61 pmol GLT-1 per milligram of membrane protein. Purification of the His-tagged protein by Ni 2C -NTA affinity chromatography enabled the routine production and purification of milligram quantities of fully functional transporter. Transport activity required reducing conditions and the addition of extra lipid throughout the purification. The apparent molecular mass of the recombinant transporter was 73 kDa or 55 kDa, corresponding to the glycosylated and non-glycosylated form, respectively. Both forms were active upon separation on a lectin column and reconstitution into liposomes. Glycosylated and non-glycosylated GLT-1 were transported to the plasma membrane with equal efficiency. Our results show that N-glycosylation does not affect the trafficking or the transport activity of GLT-1. The low-resolution structure of GLT-1 was determined by electron microscopy and single particle reconstruction. q 2005 Elsevier Ltd. All rights reserved. Keywords: glutamate transporter; expression; glycosylation; structure; electron microscopy *Corresponding author Introduction Glutamate is the major excitatory neuro- transmitter in mammalian brain. The concentration of glutamate in the synaptic cleft is regulated by secondary glutamate transporters, which maintain an extracellular concentration below neurotoxic levels. 1–5 Glutamate uptake is electrogenic, 6–8 involving co-transport of three sodium ions, a proton, and one glutamate molecule in exchange for a potassium ion. 9–11 Five eukaryotic glutamate transporters have so far been identified and cloned: GLT-1, 10 GLAST-1, 12 EAAC-1, 13 EAAT4, 14 EAAT5. 15 The structural properties of the glutamate transporters have been studied extensively by biochemical methods. 16–21 Recently, the first high- resolution structure of a glutamate transporter homologue from Pyrococcus horikoshii was reported. 22 However, there is as yet no direct structural information on the medically relevant mammalian glutamate transporters. Glutamate transporters are present at low concentrations in their native tissue, and therefore heterologous overexpression is an essential prerequisite for obtaining sufficient amounts of protein for structural, biochemical and biophysical studies. Recently, several proteins, including a number of G-protein coupled receptors, have been success- fully produced in tissue culture cells transfected with Semliki Forest virus (SFV). 23 The SFV expression system 24,25 has a number of advantages compared to other expression systems that use mammalian cell lines: (i) it produces high yields, 0022-2836/$ - see front matter q 2005 Elsevier Ltd. All rights reserved. Abbreviations used: SFV, Semliki Forest virus; ER, endoplasmic reticulum; DDM, dodecylmaltoside; SYM, (2S,4R)-4-methyl glutamate. E-mail address of the corresponding author: werner.kuehlbrandt@mpibp-frankfurt.mpg.de doi:10.1016/j.jmb.2005.06.036 J. Mol. Biol. (2005) 351, 598–613