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

The successful translocation of many secretory proteins and the integration of several transmembrane proteins have been studied using a protease protection assay. The assay is based on the analysis of polypeptides and polypeptide fragments that are protected from proteolytic degradation by the lipid bilayer of the microsomal vesicles. Since in polytopic membrane proteins, such as connexins, that traverse the membrane several times, several relatively small polypeptide fragments were expected to be protected, we combined the classical protease protection assay with the specific immunoprecipitation of protected protein fragments to obtain some information on the transmembrane organization of the processed connexin proteins. Assays were performed with/31 GJ protein since a complete set of anti-peptide antibodies recognizing all cytoplasmic and extracellular domains of the 13i GJ protein (NH:-terminal, intracellular loop, COOH terminal, and extracellular domains; Fig. 7 ) was available. Microsomes containing in vitro-translocated/31 GJ protein were incubated with different concentrations of trypsin or proteinase K (PK), respectively. Before the immunoprecipi-COOH-terminal domain), or within the membrane bilayer (transmembrane domains M1-M4) are indicated. The regions recognized by the different connexin specific anti-peptide antibodies (/5~B,/31 1-6, GAP 10, 131E, 131J, ~tJ, 13iS, and c~lS) used in this study are schematically marked. lanes 10 and 12) and mature o~-factor (a-factor*, lanes 14 and 16) , were generated only in reactions containing active SRP. (lanes 3, 6, 9, and 16) , trypsin (0.5 mg/ml, lanes 4, 7, 10, and 17, and 1 mg/ml, lanes 11, 12, and 18), or proteinase K (PK, 0.1 m~/ml, lanes 5, 8, 13, and 19, and 0.5 mg/ml, lanes 14, 15, and 20) , respectively, was added to aliquots of the membrane insertion reactions. Trypsin or PK, together with NP-40 (1% final concentration) was added to control aliquots (lanes 12 and 15). After blocking pmtease activity, GJ polypeptides and polypeptide fragments protected from proteolytic degradation were immunoprecipitated with anti-peptide antibodies specific for the NH2-terminal domain (fl~B), the extracellular loops E1 and E2 (fl~E), the intracellular loop region (B~J), and the COOHterminal domain (#~S). Immunoprecipitations were analyzed on SDS-protein gels, allowing the resolution of small polypeptide fragments. Polypeptides were visualized by fluorograpby. A linear representation of the B~ GJ protein is shown under the fluorogram. Numbers for the amino acid residues for the different topological domains corresponding in their graphical pattern to Fig. 5 B , and the binding sites for the anti-peptide antibodies are given. The locations of radioactive labeled methionine residues are indicated by diamonds.