THE TOPOGENIC FATE OF THE POLYTOPIC TRANSMEMBRANE PROTEINS, SYNAPTOPHYSIN AND CONNEXIN, IS DETERMINED BY THEIR MEMBRANE-SPANNING DOMAINS

The synaptophysins and connexins are polytopic transmembrane proteins of similar secondary structure that accumulate as multiple homo-oligom ers in specialized membrane regions, the presynaptic transmitter vesic les or gap junctions, Transfection and expression of the respective ge nes in cultured epithelial cells results in the de novo formation of e ither small cytoplasmic, synaptophysin-rich vesicles, or functional ga p junctions consisting of clustered connexin molecules. To examine the molecular requirements for the specific enrichment and topogenesis of both types of molecule, chimeric cDNAs were constructed composed of d ifferent parts of the rat synaptophysin and rat liver connexin32 genes , Expression of the encoded chimeric polypeptides in hepatocellular ca rcinoma-derived cells showed that only chimeras with all four transmem brane domains from either parent molecule were delivered to their spec ific destination. In contrast, chimeras with transmembrane domains fro m both connexin32 and synaptophysin were always retained in the endopl asmic reticulum, The topogenic nature of the transmembrane domains was further demonstrated by deletion mutagenesis, indicating that removal of cytoplasmic end domains or intravesicular loops does not abolish t argeting, On the other hand, excision of individual transmembrane doma ins or introduction of point mutations in transmembrane segments resul ted in retention in the endoplasmic reticulum.