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

To determine the intracellular distribution of the different connexin products generated under these conditions in vivo, highly induced BHK cells expressing txt or/3~ GJ protein, respectively, were fractionated into subcellular membrane fractions consisting of PM, Golgi membranes, and rough ER membranes (microsomes). The cleaved connexin products were only detectable in the ER membrane containing fractions (Fig. 8 B, lanes 1, 5, and 9 ). This result indicates that the NH2-terminally cleaved connexin product generated in the transfected BI-IK cells was similar to the product generated in the in vitro translocation assays, and for example, was not related to the degradation of PM GJ structures. A small amount of processed connexin protein present in the PM fraction prepared from/3, GJ protein expressing cells (Fig. 8 B, lane 6 ) was most likely related to a slight contamination of this subcellular fraction with some ER derived membranes. This was indicated by the presence of some PDI, an ER specific marker protein in the PM fraction (Fig. 8 C) . Only the intact connexin product was detected in the Golgi fractions ( Fig. 8 B, lanes 3, 7, and 1/) . Additional, slower migrating protein products present in Fig. 8 , A and B, (also in Fig. 3 A) represent oligomerized connexin proteins (especially in Fig. 8 A, lanes 3 and 5; Fig. 8 B, lanes 5-8; Fig. 3 A, lanes 4 and 5) and some other nonspecifically detected cellular proteins (especially in Fig. 8 B, lanes 1-4 and 9; Fig. 3 A, lane 3) .