AN INVESTIGATION OF THE MEMBRANE TOPOLOGY OF THE IONOTROPIC GLUTAMATE-RECEPTOR SUBUNIT GLUR1 IN A CELL-FREE SYSTEM

We have utilized cell-free translation in rabbit-reticulocyte lysate s upplemented with canine pancreatic microsomal membranes to study the p rocessing and membrane topology of the rat ionotropic glutamate recept or subunit GluR1. In vitro-synthesized RNA encoding GluR1 was translat ed to yield a primary translation product with an apparent molecular m ass of 99 kDa. In the presence of microsomal membranes this protein wa s processed to give a band of 107 kDa. Treatment with peptide-N-glycos idase F showed that this increase in molecular mass was due to N-linke d glycosylation. Incubation of the processed receptor with proteinase K revealed the presence of a 68 kDa protease-resistant band which decr eased to 56 kDa following deglycosylation. A deletion mutant (GluR1M1) lacking the predicted transmembrane domains was fully translocated ac ross the microsomal membrane and protected from the action of the prot ease. The mutant and wild-type receptor could be immunoprecipitated by anti-peptide antibodies directed against the C-terminus. Following tr anslocation of the wild-type and mutant receptor across the microsomal membrane and treatment with proteinase K the antibody binding to GluR 1 was abolished, but was retained for GluR1M1. These data allow identi fication of the orientation of the N- and C-termini of GluR1 within th e microsome; results which are consistent with an extracellular N-term inal and intracellular C-terminal localization following incorporation into the plasma membrane.