Topology of the Mr 27,000 liver gap junction protein. Cytoplasmic localization of amino- and carboxyl termini and a hydrophilic domain which is protease-hypersensitive.
Hydropathy analysis of the Mr 27,000 rat liver gap junction protein sequence deduced from a cDNA clone has suggested the presence of four transmembrane segments (Paul, D. L. (1986) J. Cell Biol. 103, 123-134). In the present report, several features of the molecular topology of the protein were investigated by microsequence analysis of peptides generated by treatment of isolated gap junctions with a variety of proteases. Under the experimental conditions used, the proteases had access only to the portion of the Mr 27,000 protein that was originally (in vivo) the cytoplasmic surface of the gap junction. Microsequencing of the peptides resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicates that the amino terminus of the protein is disposed at or near the cytoplasmic surface of the gap junction, and that this surface also contains a protease-hypersensitive hydrophilic sequence between residues 109 and 123, presumably connecting the second and third transmembrane segments. Immunocytological localization of binding of monoclonal antipeptide antibodies demonstrates that the carboxyl terminus of the protein is also localized to the cytoplasmic surface of the gap junction. No protease sensitivity was found in the hydrophilic sequences thought to connect either the first and second transmembrane segments or the third and fourth segments, supporting the model's prediction that these sequences face the narrow intercellular gap which cannot be penetrated by proteases.
Hertzberg
E L
EL
Verna and Marrs McLean Department of Biochemistry, Baylor College of Medicine, Houston, Texas 77030.
Disher
R M
RM
Tiller
A A
AA
Zhou
Y
Y
Cook
R G
RG
D000595:Amino Acid Sequence; D000818:Animals; D016282:Aspartic Acid Endopeptidases; D017630:Connexins; D003593:Cytoplasm / Q000032:analysis; D004591:Electrophoresis, Polyacrylamide Gel; D010450:Endopeptidases / Q000378:metabolism; D005455:Fluorescent Antibody Technique; D007150:Immunohistochemistry; D008565:Membrane Proteins / Q000032:analysis*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D051381:Rats
D017630:Connexins; D008565:Membrane Proteins; D010450:Endopeptidases; C022325:aspartic proteinase A; D016282:Aspartic Acid Endopeptidases