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

To obtain some information on the transmembrane organization of the cleaved connexin polypeptides, protease protection assays (Morimoto et al., 1983; Scheele, 1983) were used in this study in combination with the immunoprecipitation of protein fragments protected from degradation. In previous studies, this protease protection assay has been used extensively to analyze the membrane insertion of proteins and to determine the membrane orientation of different types of membrane anchored proteins (Lipp and Dobberstein, 1986b; Spiess and Lodish, 1986; Zerial et al., 1986; Mayer et al., 1988; Chavez and Hall, 1992; Skach et al., 1994; Gafvelin and yon Heijne, 1994) . We found that the intracellular loop domain of the/31 connexin was protected from proteolytic degradation when trypsin or proteinase K were added exogenously to the microsomes, while it was degraded when the microsomes were permeabilized with detergent before the addition of protease (Fig. 7) . This result indicates that either the intracellular loop domain was located inside the vesicle lumen, suggesting an inverted membrane topology for the NH2-terminaily processed connexins (hypothesized in an earlier report, Falk et al., 1994) , or that the intracellular loop was resistant to proteolytic degradation under the conditions applied. An inverted membrane topology would be consistent with the processing of a cryptic signal peptidase cleavage site motif (small, uncharged amino acid residues in positions -3 and -1; von Heijne, 1983) that exists in connexins in front of transmembrane region M1. Proteolytic processing at this site is suggested by the mobility shift of 2-2.5 kD observed on the protein gels.