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

that the pancreatic microsomes serve as a competent recipient in vivo for unprocessed full length connexins. Although oriented with their amino terminus in the cytoplasm, the analysis of the cleavage reaction indicated that an unprecedented processing by signal peptidase resulted in the removal of an amino-terminal portion of the cormexins. Variable amounts of similar cormexin cleavage products were also identified in the ER membranes of connexin overexpressing cells. The amount generated correlated with the level of protein expression. These results demonstrate that the connexins contain a cryptic signal peptidase cleavage site that can be processed by this enzyme in vitro and in vivo in association with their membrane insertion. Consequently, a specific factor or condition must be required to prevent this aberrant processing of connexins under normal conditions in the cell. C rIANNEL-forming membrane proteins, similar to other plasma membrane (PM) t proteins, are usually synthesized on ribosomes bound to the ER membrane and inserted cotranslationally into this membrane. Subsequently, the proteins are transported to the PM passing through the classical secretory pathway of the cell (Pfeffer and Rothman, 1987) . For translocation into the ER membrane, the proteins need a signal specific for ER membrane targeting that is encoded in the amino acid sequence of the polypeptide chain, either as a cleavable signal sequence on the NH~ terminus and processed by signal peptidase, or as an internal signal anchor sequence (Blobel, 1980; Walter et al., 1984) . After insertion into the ER membrane, the polypeptide domains fold into their final conformation, and they eventually oligomerize most likely with the help of different chaperones, folding enzymes, and metabolic energy (Rothman, 1989; Hendrick and Hartl, 1993) . It can be concluded that connexins follow the secretory pathway based on subcellular connexin distribution studies presented in this paper and from studies reported by other investigators. Studies performed with wild-type connexin-expressing cells isolated from rat liver (this study; Rahman et al., 1993) and dog pancreas (acinar cells, this study), as well as tissue culture cell lines naturally expressing connexins (normal rat kidney fibroblasts [NRK 49F], Musil and Goodenough, 1991) , or transfected with connexin cDNAs (BHK cells; this study) showed connexin proteins in the ER membranes, the Golgi membranes, and in the plasma membranes isolated from these cells.