Signal recognition particle-dependent membrane insertion of mouse invariant chain: a membrane-spanning protein with a cytoplasmically exposed amino terminus

Methods mRNA Purification and Size Fractionation. Total mRNA was isolated from the spleens of SL2 mice by a modified guanidinium hydrochloride method (10) . Approximately 20 g of frozen spleens were homogenized in 200 ml 6 M guanidinium HCI/1 mM dithiothreitol/20 mM NaAc pH 7.0 (buffer I), using a Sorvall Omni-mixer at full speed for pulses of 30-s each. The homogenate was centrifuged for 5 rain at 5,000 g at 4"C. The resulting supernatant was adjusted to pH 5.0 with 4 N acetic acid and the RNA was precipitated by adding half a volume of ice-cold ethanol. Insoluble material was collected by centrifugation and dissolved in 100 ml of buffer 1, adjusted to pH 5, and reprecipitated as above. After centrifugation, the pellet was dissolved in 10 ml 50 mM Tris/HCl, pH 7.5, 120 mM NaCl, 5 mM EDTA, 1% SDS (buffer 2), and extracted twice with phenol/chloroform. Poly (A) + RNA was obtained by affinity chromatography on an oligo(dT)-cellulose column, mRNA was size fractionated by sucrose gradient centrifugation. Fractions enriched in mRNA that code for Ii chain were identified by cell free translation (see below) and immunoprecipitation (12) .