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

PMs were prepared from rat liver and dog pancreas using the method described by Stauffer et al. (1991) with the following modifications. One rat liver and '~20 g of a dog pancreas were used per preparation, respectively. 6,000 g pellets were resuspended in 5 ml buffer, and 10 ml of 67% (wt/wt) sucrose was added. Samples were placed in one tube each, overlayed with 25 ml of 30% (wt/wt) sucrose and centrifuged at 27,000 rpm for 105 rain in a rotor (SW 28; Beckman Instruments, Inc.) PM were collected from the interphase of the gradients, and quick-frozen in liquid nitrogen before further analysis. Rough ER membranes were prepared from rat liver and dog pancreas following the method described by Walter and Biobel (1983) . Before immunoblot analysis, GJ protein was enriched from 200-~1 aliquots of the RER membrane (microsomes) containing fraction and from the in vitro translation reactions analyzed in Fig. 3 A by immunoprecipitation, as described above./3t GJ protein-specific monoclonal antibody M12.13 (Goodenough et al., 1988) , reacting with the ~LJ-peptide, was purified from mouse ascites and covalently coupled to hydrazine-activated agarose (Afli-Gel HZ; Bio Rad Lab, Hercules, CA), following the instructions of the manufacturer. Precipitated proteins and aliquots of the PM and lysate fraction were then subjected to SDS-PAGE and transferred to a nitrocellulose membrane for immunoblot analysis with ~lJ antibodies. Detection of bound antibody was performed by coupled chemiluminescence (Amersham Corp.).