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

In previous studies, coupled in vitro translation/membrane Figure 1 . In vitro translation in the absence and in the presence of microsomes. Synthetic RNAs coding for GJ proteins cd,/3~,/32,/~3, and a3 were translated in the rabbit reticulocyte lysate system in the absence (-) or presence (+) of canine pancreatic micmsomes for the indicated time periods (t in minutes). Fluorographs of the translation products after SDS-PAGE are shown. Full-size polypeptides are indicated as c~1,/~t,/3z,/33, and a3. In reactions including microsomes, a second connexin-specific translation product was generated and marked as a~',/~1',/~z',/~3', and c~3' . Translation reactions showed the first detectable synthesis of full-size GJ protein after ,07 min in the presence of microsomes (shown for cq GJ protein, lane 6). The faster migrating GJ protein product is generated after the same translation time. Aggregates of GJ polypeptides not entering the separating gels were especially prominent after long reaction times and in absence of microsomes. A schematic representation of the overall membrane topology for cormexins is shown on the right. The connexins mainly differ from each other in the length of their COOH-terminal cytoplasmic domain. The cytoplasmic and the lumenal/exoplasmic sides of translocation assays supplemented with canine pancreatic microsomes (Walter and Blobel, 1983) have been found to integrate several type I, II, and III membrane proteins with a relevant membrane topology (e.g., Lipp and Dobberstein, 1986b; Spiess and Lodish, 1986; Zerial et al., 1986; Mayer et al., 1988) . The microsomes, which are vesicles derived from the ER membranes of pancreatic acinar ceils during isolation, are believed to contain all ER lumenal and membrane proteins necessary for the successful translocation of secretory and membrane anchored proteins (Nicchitta and Blobel, 1993) .