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-oligomers in specialized membrane regions, the presynaptic transmitter vesicles or gap junctions. Transfection and expression of the respective genes in cultured epithelial cells results in the de novo formation of either small cytoplasmic, synaptophysin-rich vesicles, or functional gap 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 different parts of the rat synaptophysin and rat liver connexin32 genes. Expression of the encoded chimeric polypeptides in hepatocellular carcinoma-derived cells showed that only chimeras with all four transmembrane domains from either parent molecule were delivered to their specific destination. In contrast, chimeras with transmembrane domains from both connexin32 and synaptophysin were always retained in the endoplasmic 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 targeting. On the other hand, excision of individual transmembrane domains or introduction of point mutations in transmembrane segments resulted in retention in the endoplasmic reticulum.
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Division of Cell Biology, German Cancer Research Center, Heidelberg.
D000818:Animals; D001483:Base Sequence; D002451:Cell Compartmentation; D002460:Cell Line; D003001:Cloning, Molecular; D017630:Connexins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017931:DNA Primers / Q000235:genetics; D018076:DNA, Complementary / Q000235:genetics; D004721:Endoplasmic Reticulum / Q000378:metabolism; D007425:Intracellular Membranes / Q000378:metabolism; D016253:Microscopy, Immunoelectron; D008969:Molecular Sequence Data; D016296:Mutagenesis; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016708:Synaptophysin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000097002:Gap Junction beta-1 Protein
D017630:Connexins; D017931:DNA Primers; D018076:DNA, Complementary; D011993:Recombinant Fusion Proteins; D016708:Synaptophysin; D000097002:Gap Junction beta-1 Protein