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

BHK cells were split into 10-cm dishes and grown overnight in DME to reach semiconfluence. Medium was replaced by methionine-free DME (Gibeo Life Technologies, Grand Island, NY), 150 #Ci Tran35S-label and CaC12 was added from a 50-mM stock solution to the concentrations indicated, and cells were labeled for 8-12 h. Cells were either lysed directly in RIPA buffer and processed for immunoprecipitation, or processed further to prepare subcelluiar membranes. Subcellular membrane fractions were prepared by the method of Bole et al. (1986) with the following modifications. Cells were chilled on ice and scraped from the plate in 1 ml PBS containing 0.25 M sucrose, and then dispersed with 10-20 strokes in a tightfitting dounce homogenizer. Opaque bands at the interfaces of the sucrose step gradient, containing either Golgi membranes, PM, or rough ER membranes, respectively, were harvested using a needle and syringe. Aliquots of the fractions were added to RIPA buffer and processed for immunoprecipitation, or aiiquots were directly subjected to SDS-PAGE and transferred to nitrocellulose membranes for immunoblot analysis with a PDI-specific antibody, as described above. Kumar and Gilula [1992] ) were cloned into the transcription vector pSP64T, and synthetic RNA was transcribed using SP6 polymerase CKrieg and Melton, 1984) . This strategy allowed the synthesis of connexin-specific cRNAs that were translated much more efficiently than the natural connexin RNAs in competent cell lysates, such as rabbit reticulocyte lysates or wheat germ extracts. Resulting connexin translation products generated in the absence of microsomes corresponded to the molecular masses for these proteins as predicted from their amino acid sequences (Fig. 1, lanes 1 , 2, 11, 12, 15, 16, 19 , and 2/). Identification of the translation products by specific anti-peptide antibodies directed against different regions of the connexins (see below) provided additional confirmation for the synthesis of full-length connexin proteins.