Mucin-like domain of enteropeptidase directs apical targeting in Madin-Darby canine kidney cells.
Enteropeptidase, a type II transmembrane protein of the enterocyte brush border, is sorted directly to the apical membrane of Madin-Darby canine kidney II cells. Apical targeting appears to be mediated by an N-terminal segment that contains a 27-amino acid residue O-glycosylated mucin-like domain consisting of two short mucin-like repeats, A and B. Targeting signals within these repeats were characterized by using green fluorescent protein (GFP) as a reporter. Constructs with a cleavable signal peptide and both repeats A and B were secreted apically. Similar constructs lacking mucin repeats were secreted randomly. Either repeat A or B was sufficient to direct apical targeting of GFP. O-linked oligosaccharides alone were not sufficient for targeting because fusion to a different O-glycosylated motif did not alter the random secretion of GFP, and several constructs with mutations in either repeat A or B were O-glycosylated and secreted randomly. In addition, repeat B appears to contain an apical targeting signal that functions in the absence of glycosylation. Density gradient centrifugation indicated that, unlike several other apically targeted membrane and soluble proteins, apical sorting of mucin-GFP chimeric proteins does not appear to utilize lipid rafts.
Zheng
Xinglong
X
Department of Pathology and Immunology, Washington University School of Medicine, 660 S. Euclid Avenue, St. Louis, MO 63110, USA.
Sadler
J Evan
JE
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000596:Amino Acids; D000818:Animals; D002460:Cell Line; D002499:Centrifugation, Density Gradient; D004285:Dogs; D006026:Glycoside Hydrolases / Q000378:metabolism; D006031:Glycosylation; D049452:Green Fluorescent Proteins; D007668:Kidney / Q000378:metabolism; D008164:Luminescent Proteins / Q000378:metabolism; D021962:Membrane Microdomains / Q000378:metabolism; D008969:Molecular Sequence Data; D009077:Mucins / Q000737:chemistry*; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010957:Plasmids / Q000378:metabolism; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012694:Serine / Q000737:chemistry; D015398:Signal Transduction; D013912:Threonine / Q000737:chemistry; D013997:Time Factors; D014162:Transfection
D000596:Amino Acids; D008164:Luminescent Proteins; D009077:Mucins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D013912:Threonine; D012694:Serine; D006026:Glycoside Hydrolases