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

Performing translocation reactions with variable concentrations of microsomes showed that the amount of modified, faster migrating connexin protein was directly related to the concentration of microsomes in the translocation reactions, eventually reaching 100% completion (Fig. 2) . In reactions containing low concentrations of microsomes (0.05-0.5 Eq/10/~1 reaction volume, Fig. 2, lanes 2-6and 12-16) , only a small portion of the translation product was processed, while in reactions containing high microsome concentrations (1.5-2 Eq/10/~1 reaction volume, Fig. 2 , lanes 9 and 10, 19 and 20), the faster migrating connexin product was almost the only detectable product generated. Approximately equal amounts of processed and unprocessed connexins were generated when intermediate membrane concentrations ('~0.75-1 Eq/10/~1 reaction volume, Fig. 2 , lanes 7and 8, 17and 18) were used in the experiments. Translocation reactions in all subsequent experiments (and in the experiments shown in Fig. 1 ) were carried out with intermediate membrane concentrations ('M Eq/10 #1 reaction volume), leading to the generation of complete and modified connexin proteins that appear as a double-band pattern on the fluorograms (a/a' and/3/13' GJ protein).