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

tation of protease-protected connexin fragments, protease activity was blocked following the method described by Chavez and Hall 0992). Protected protein fragments were analyzed by immunoprecipitation, and the protected fragments were separated on special SDS gels (Thomas and Kornberg, 1975) . No connexin fragments were immunoprecipitated after protease treatment using antibodies directed against the NH2-terminal region (BIB antibodies; Fig. 7, lanes 4 and 5) or the COOH-terminal region (B~S antibodies; Fig. 7, lanes 17-20) ; both regions should be located predictably outside of the microsomes and exposed to the proteases. Furthermore, no connexin fragments were immunoprecipitated using antibodies directed against the extraceUular loops E1 and E2 (fl~E antibodies; Fig. 7, lanes 7 and 8) that should be located predictably in the lumen of the microsomes. This could be caused by the low abundance of these fragments and/or the relatively low affinity of the /31E antibodies (compare the amount of B, protein immunoprecipitated with time antibodies (Fig. 7, lane 6) with that precipitated with fl~J or fitS antibodies (Fig. 7, lanes 9 and 16) . On the other hand, antibodies directed against the intracellular loop of ~ protein (/31J antibodies) that should be located predic-tably outside of the microsomes immunoprecipitated labeled /3t polypeptide fragments of apparent sizes of ,'~9-10 and 15-17 kD that were protected from proteolysis by both proteases (Fig. 7, lanes 10, 11, 13, and 14) . However, degradation of the intracellular loop was observed when detergent (1% NP-40, final concentration) was added before the addition of trypsin or PK (Fig. 7, lanes 12 and 15) . No labeled protein fragments were immunoprecipitated under these conditions after trypsin treatment (Fig. 7, lane 12) , and only a small polypeptide fragment with an apparent molecular mass of ~4 kD was resistant to PK digestion (Fig. 7, lane 15 ). /~ GJ protein that was not exposed to proteases was precipitable with all the individual antibodies under the conditions used (Fig. 7, lanes 3, 6, 9, and 16) .