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

Various control experiments exclude the possibility that the processing results from the cloning strategy (the same processing occurred with connexin cRNAs derived from other in vitro transcription vectors), the translation system (reticulocyte lysates vs wheat germ extracts, Fig. 1 vs Fig. 5 ), the microsomes (five membrane batches prepared independently from individual canine pancreas were used), or the reaction conditions that were applied. Different control proteins (membrane anchored: aeetylcholine receptor ~7 subunit; secretory: prolactin, yeast a factor; cytoplasmic: /3-globin; Fig. 4 ) translated in parallel to the connexin proteins showed the expected results described previously for these proteins. Reaction conditions were modified from more reducing conditions (presence of DTT) to oxidizing conditions (~5 mM final concentration oxidized glutathione was added) to allow S-S bridge formation in the newly syn- Figure 3 . Connexins isolated from natural sources correspond to the full-size connexins synthesized in vitro. (A) PM and PER membranes were isolated from rat liver and dog pancreas acinar cells and native 31 GI protein, endogenous to these membranes, was analyzed by immunoblot analysis using a connexin specific antibody as described in Materials and Methods. Complete cell lysates (lanes I and 4) and/31 GJ protein synthesized in vitro in the absence (-) or presence (+) of microsomes (lanes 7and 8) were analyzed in parallel. The RER membrane fraction isolated from dog pancreas 0ane 6) is identical to the microsomal membranes used in the in vitro translocation assays. (B) Determination of alkaline PDE activity in subcellular fractions and cell lysates prepared from dog pancreas. The samples contained similar protein concentrations. Three independent preparations were analyzed. The activity of the PM marker enzyme was very low in the RER membranes containing fractions, indicating that the eonnexin detected in these fractions originated from the ER membranes of the pancreatic cells.