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

The translocation of connexins into the ER membrane was analyzed in vivo to determine if the observed signal peptidase processing is related to the normal membrane translocation process of connexins in vivo. Since the amount of connexin polypeptides present in the ER membranes is relatively low in normal connexin expressing cells (rat hepatocytes, dog pancreatic acinar cells, Fig. 3 ), making such an analysis relatively difficult, we expressed higher levels of the ot~ and/~ GJ proteins for this analysis by using heterologous eukaryotic protein expression systems (yeast, baculovirus, BHK cells, Fig. 8 A) . Expressed connexins were analyzed either by immunoblot analyses or by the immunoprecipitation of radiolabeled proteins. Analysis of the expressed proteins showed that connexins with a processed NH2-terminal domain, similar to the products found in vitro, were detectable in all cell types analyzed (Fig. 8 A, left panel: ~1 GJ protein-transfected yeast cells, lanes 1, 3, 5; middle panel: insect cells infected with an Otl GJ protein expressing baculovirus, lanes 10 and 11; right panel: BHK cells stably transfected with c~ or/3~ GJ protein, lanes 18, 19, 25, 27, 29, and 30) . While yeast transfectants generated nearly equal amounts of processed and full-size connexins, the majority of the connexin protein generated in the infected insect cells, and especially in the transfected BHK cells, appeared to be the unprocessed full-size protein. Using the transfected BHK cells that allow the expression of variable amounts of protein (the connexin cDNAs are inserted behind a heavy metal inducible promoter), it was possible to demonstrate that the generation of the NH~-terminally processed cormexin products correlated with the level of connexin expression (Fig. 8 A, right panel) . Under moderate expression conditions (50 #M zinc), only intact c~t or/3t GJ protein was detectable (Fig. 8 A, lanes 14-16, and 22 and 23) , while conditions of intermediate (100 #M zinc, Fig. 8 A, lanes 24 and 25) or high connexin protein expression (125-150 #M zinc, Fig. 8 A, lanes 17-19 and 26--30) resulted in the synthesis of increasing amounts of NH2-terminally cleaved (c~1' and/30, as well as intact cormexin proteins.