Apical sorting of bovine enteropeptidase does not involve detergent-resistant association with sphingolipid-cholesterol rafts

Enteropeptidase is a heterodimeric type II membrane protein of the brush bo rder of duodenal enterocytes. In this location, enteropeptidase cleaves and activates trypsinogen, thereby initiating the activation of other intestin al digestive enzymes. Recombinant bovine enteropeptidase was sorted directl y to the apical surface of polarized Madin-Darby canine kidney cells. Repla ce ment of the cytoplasmic and signal anchor domains with a cleavable signa l peptide (mutant proenteropeptidase lacking the amino-terminal signal anch or domain (dSA-BEK)) caused apical secretion. The additional aminoterminal deletion of a mucin-like domain (HL-BEK) resulted in secretion both apicall y and basolaterally, Further deletion of the noncatalytic heavy chain (L-BE K) resulted in apical secretion. Thus enteropeptidase appears to have at le ast three distinct sorting signals as follows: the light chain (L-BEK) dire cts apical sorting, addition of most of the heavy chain (HL-BEK) inhibits a pical sorting, and addition of the mucin-like domain (dSA-BEK) restores api cal sorting. Inhibition of N-linked glycosylation with tunicamycin or disru ption of microtubules with colchicine caused L-BEK to be secreted equally i nto apical and basolateral compartments, whereas brefeldin A caused basolat eral secretion of L-BEK. Full-length BEK was not found in detergent-resista nt raft domains of Madin-Darby canine kidney cells or baby hamster kidney c ells. These results suggest epical sorting of enteropeptidase depends on N- linked glycosylation of the serine protease domain and an amino-terminal se gment that includes an O-glycosylated mucin-like domain and three potential N-glycosylation sites. In contrast to many apically targeted proteins, ent eropeptidase does not form detergent-resistant associations with sphingolip id-cholesterol rafts.