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

In this study, we analyzed the translocation and the transmembrane organization of the gap junction (GJ) proteins (connexins) in the membranes of the ER. Connexins, together with other aqueous channel-forming membrane proteins, represent specialized polytopic transmembrane proteins that are distinguished by the localization of charged amino acid residues within their transmembrane regions. At present, 12 different connexins have been cloned and sequenced from mammals. All represent structurally conserved, nonglycosylated members of a multigene family (Kumar and Gilula, 1992) . Connexins traverse the PM bilayer four times oriented with their amino and carboxy termini located in the cytoplasm (Ncyt-C¢~ orientation). This membrane topology was suggested by hydropathy analyses of the primary amino acid sequences (Milks et al., 1988) , and it is supported by various topological analyses of the connexin protein structure, such as site-directed antibody localizations combined with immunoelectron microscopy (Hertzberg et al., 1988; Milks et al., 1988; Laird and Revel, 1990; Rahman and Evans, 1991; E1 Aoumari et al., 1992) , protease digestions of isolated GJ structures (Zimmer et al., 1987; Hertzberg et al., 1988) , or the analysis of intramolecular disulfide bridges (Rahman and Evans, 1991) . The NH2terminal cytoplasmic domain of the connexins consists of a short stretch of 22-23 amino acid residues, and no cleavable signal sequence is present within the NH2-terminal region. Consequently, the first and/or one of the other transmembrane hydrophobic regions must function as an internal signal anchor sequence(s). After their membrane integration, connexins must oligomerize to form a functional GJ hemichannel (Makowski et al., 1977) . At the PM, two hemichannels, one provided by each of two neighboring cells, associate to form a double-membrane hydrophilic channel that creates a cytoplasmic continuity.