High-yield expression, reconstitution and structure of the recombinant, fully functional glutamate transporter GLT-1 from Rattus norvegicus.
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 x 10(6) functional transporters per cell, or 61 pmol GLT-1 per milligram of membrane protein. Purification of the His-tagged protein by Ni2+-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.
Raunser
Stefan
S
Department of Structural Biology, Max-Planck-Institute of Biophysics, 60439 Frankfurt am Main, Germany.
Haase
Winfried
W
Bostina
Mihnea
M
Parcej
David N
DN
Kühlbrandt
Werner
W
D000818:Animals; D001704:Biopolymers; D002460:Cell Line; D006224:Cricetinae; D004591:Electrophoresis, Polyacrylamide Gel; D027342:Excitatory Amino Acid Transporter 2 / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D011487:Protein Conformation; D011499:Protein Processing, Post-Translational; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification
D001704:Biopolymers; D027342:Excitatory Amino Acid Transporter 2; D011994:Recombinant Proteins