Involvement of detergent-insoluble complexes in the intracellular transport of intestinal brush border enzymes.
A number of transmembrane digestive enzymes of the porcine small intestinal brush border membrane were found to be partially Triton X-100-insoluble at 0 degree C and colocalized in gradient centrifugation experiments with the GPI-anchored alkaline phosphatase in low-density, detergent-insoluble complexes commonly known as glycolipid "rafts". Thus, aminopeptidase N (EC 3.4.11.2), aminopeptidase A (EC 3.4.11.7), dipeptidyl peptidase IV (EC 3.4.14.5), and sucrase-isomaltase (EC 3.2.1.48-10) were 34-48% detergent-insoluble. Maltase-glucoamylase (EC 3.2.1.20) was markedly less detergent-insoluble (20%), and lactase-phlorizin hydrolase (EC 3.2.1.23-62) was essentially fully soluble in detergent. In radioactively labeled, mucosal explants, the newly synthesized brush border enzymes began to associate with detergent-insoluble complexes while still in their transient, high mannose-glycosylated form, and their insolubility increased to that of the steady-state level soon after they achieved their mature, complex glycosylation, i.e., after passage through the Golgi complex. Detergent-insoluble complexes isolated by density gradient centrifugation were highly enriched in brush border enzymes, and the enrichment was apparent after only 1 h of labeling, where aminopeptidase N, sucrase-isomaltase, and alkaline phosphatase together comprised 25-30% of the total labeled, detergent-insoluble proteins, showing that sorting of newly made brush border membrane proteins into the glycolipid "rafts" does take place intracellularly. I therefore propose that, in the enterocyte, the brush border enzymes are targeted directly from the trans-Golgi network toward the apical cell surface.
Department of Medical Biochemistry and Genetics, Panum Institute, University of Copenhagen, Denmark.
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Danielsen
D000626:Aminopeptidases / Q000378:metabolism*; D000818:Animals; D001692:Biological Transport; D002499:Centrifugation, Density Gradient; D006017:Glycolipids / Q000378:metabolism; D006056:Golgi Apparatus; D007421:Intestine, Small / Q000378:metabolism*; D008871:Microvilli / Q000378:metabolism*; D017830:Octoxynol / Q000494:pharmacology*; D009834:Oligo-1,6-Glucosidase / Q000378:metabolism*; D009924:Organ Culture Techniques; D013552:Swine
D006017:Glycolipids; D017830:Octoxynol; D009834:Oligo-1,6-Glucosidase; D000626:Aminopeptidases