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

However, a proteolytic processing of the connexin proteins by signal peptidase is indicated by the results in this study. All the following properties of the connexin processing are indicative of a cleavage by signal peptidase. First, the connexins were proteolytically processed in proximity to their NH2 terminus, removing the NH2-terminal portion of the proteins. This processing appears very similar to the cleavage of cleavable ER target signal sequences (Blobel, 1980; Walter et al., 1984) . Second, the cleavage occurred concomitant with the insertion into the ER membrane, also typical for the processing of cleavable signal sequences (Fig. 1) . Third, the proteolytic cleavage reaction was restricted to the lumen of the ER vesicles (Fig. 6 A) . Signal peptidase is known to be present and active in the microsomes used in our studies (Evans et al., 1986) . Finally, the cleavage was not affected by efficient serine protease inhibitors (Fig. 6 B , DFP, TPCK, and TLCK), a property described for signal peptidase and attributed to its unusual cleavage mechanism (Daibey and yon Heijne, 1992) . Taken together, a potential proteolytic processing of connexin proteins by signal peptidase is indicated by the findings in this study, raising the question, what prevents this cleavage in vivo? Some examples of an internal signal peptidase processing on "cryptic sites were previously reported for type I (cytochrome P 450) and type II membrane proteins (invariant chain [I'y], asialoglycoprotein receptor H1 subunit) after modifying the structure of the NH2-terminal domains of those proteins that altered their overall charge (Lipp and Dobberstein, 1986a; Schmid and Spiess, 1988; Szczesna-Skorupa et al., 1988) . However, these examples clearly differ from our findings in that the connexins were translated as noumutated, wild-type proteins.