Constitutive apical secretion of an 80-kD sulfated glycoprotein complex in the polarized epithelial Madin-Darby canine kidney cell line.
The biosynthesis, processing, and apical secretion of a group of polypeptides (Kondor-Koch, C., R. Bravo, S. D. Fuller, D. Cutler, and H. Garoff. 1985. Cell. 43:297-306) are studied in MDCK cells using a specific polyclonal antiserum. These polypeptides are synthesized as a precursor protein which has an apparent Mr of 65,000 in its high mannose form. This precursor is converted into a protein with an apparent Mr of 80,000 containing complex carbohydrates and sulfate. After intracellular cleavage of the 80-kD protein, the 35-45-kD subunits are secreted as an 80-kD glycoprotein complex (gp 80) linked together by disulfide bonds. Secretion of the protein complex occurs by a constitutive pathway at the apical surface of the epithelial monolayer. Since the immediate post-translational precursor, the 65-kD protein, is hydrophilic in nature as shown by its partitioning behavior in a phase-separated Triton X-114 solution, gp 80 is segregated into the apical exocytotic pathway as a soluble molecule. The proteolytic maturation of gp 80 is blocked in the presence of chloroquine and its secretion is retarded. The 80-kD precursor is released at the apical cell surface, demonstrating that proteolytic processing is not necessary for the apical secretion of this protein. If N-glycosylation is inhibited by tunicamycin treatment the protein is secreted in equal amounts at both cell surfaces, indicating a role of the carbohydrate moieties in the vectorial transport of this protein.
Abt. Molekulare Genetik, Universität Frankfurt, Federal Republic of Germany.
J
J
Urban
K
K
Parczyk
A
A
Leutz
M
M
Kayne
C
C
Kondor-Koch
D000818:Animals; D002460:Cell Line; D002738:Chloroquine / Q000494:pharmacology; D004285:Dogs; D006023:Glycoproteins / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D007668:Kidney; D007700:Kinetics; D008715:Methionine / Q000378:metabolism; D008970:Molecular Weight; D013431:Sulfates / Q000378:metabolism; D013462:Sulfur Radioisotopes; D013464:Sulfuric Acids / Q000032:analysis*
D006023:Glycoproteins; D013431:Sulfates; D013462:Sulfur Radioisotopes; D013464:Sulfuric Acids; D002738:Chloroquine; D008715:Methionine