Membrane insertion of gap junction connexins: polytopic channel forming membrane proteins

All plasmids used for in vitro transcription were linearized, phenolchloroform extracted, and ethanol precipitated. Transcription reactions were performed with a Riboprobe transcription kit (Promega Biotech). In vitro translation reactions were performed in nuclease-treated rabbit reticulocyte lysate or wheat germ extracts supplemented with [35S]methionine (Amersham Corp., Arlington Heights, IL). Reactions were generally performed for 30-60 min at 32°C and 28°C, respectively. Canine pancreas rough microsomal membranes or salt-extracted microsomal membranes were generally present at a final concentration of 1 Eq/10/~l of translation reaction. Purified SRP was used at a final concentration of 10 U/10/~1 reaction volume. Canine pancreas rough microsomes were prepared as described by Walter and BIobel (1983) . To test the influence of protease inhibitots on the proteolytic processing of cormexins, all components of the translocation assays were mixed together except RNA. Protease inhibitors N-tosyl-L-phenylalanine chloromethyl ketone (TICK), Nt~-p-tosyl-lysine chloromethyl ketone (TLCK), diisopropyl fluorophosphate (DFP), E-64, and lenpeptin were added to individual aliquots to a final concentration of 1 mM from 10x stock solutions and incubated for 10 re_in at 30°C before synthetic RNA was added. To modify reaction conditions from more reducing conditions (small amounts of DTT are present in the [35S]methionine and in the microsome preparations) to oxidizing conditions that allow posttranslational S-S bridge formation, oxidized glutathione was added in variable amounts from 0.1 to 5 mM final concentration to the translocation reactions, according to Marquardt ct al. (1993) .