Biosynthesis of intestinal microvillar proteins. Evidence for an intracellular sorting taking place in, or shortly after, exit from the Golgi complex

Abstract

Pig small intestinal mucosal explants, labelled with [35S]-methionine, were fractionated into Mg2+-precipitated (intracellular and basolaterdl) and microvillar membranes, and the orientation of newly synthesized aminopeptidase N (EC 3.4.1 1.2) in vesicles from the two fractions was studied by its accessibility to proteolytic cleavage. The mature polypeptide of M , 166000 from the latter fraction was cleaved by trypsin, proteinase K and papain, consistent with an extracellular location of the enzyme at its site of function. In contrast, both the mature form and the transient form of M , 140000 from the Mg2+-precipitated fraction were equally well protected from proteolytic cleavage (in the absence of Triton X-100). This indicates that the basolateral plasma membrane is unlikely to be involved in the post-Golgi transport of newly synthesized aminopeptidase N and suggests instead a direct delivery of the enzyme to the apical plasma membrane. A crude membrane preparation from labelled explants was used in immunoelectrophoretic purification of membranes to determine at what stage during intracellular transport newly synthesized microvillar enzymes are sorted, i. e., accumulated in areas of the membrane from where other proteins are excluded. The transient form of aminopeptidase N was only moderately enriched by immunopurification, using antibodies against different microvillar enzymes, but the mature form was enriched approximately 30-fold from explants, labelled for 30 min. This suggets that for microvillar enzymes, the aspects of sorting studied take place in, or shortly after exit from, the Golgi complex.

Alterations in the establishment and maintenance of epithelial cell polarity as a basis for disease processes.

“…1). Similar results have been obtained in studies of protein targeting in the small intestine (32), although conflicting reports using a similar model, in the small intestine, have been reported by two different laboratories (33,34). Clearly, a basolateral to apical membrane "tract" exists as hepatocytes, enterocytes, and transfected MDCK cells use this transport pathway for the transcytosis of polymeric Igs (35).…”

Chapter 2 Biogenesis and Sorting of Plasma Membrane Proteins

“…While all of these results are extremely interesting, cell fractionation studies are always open to the criticism that difficult-to-control-for and difficult-to-measure cross-contamination may artifactually skew the results. Similar experiments in other laboratories suggest that microvillar proteins are vectorially sorted in intestinal cells (Danielsen and Cowell, 1985).…”

Epithelial cell polarity as reflected in enterocytes

“…Based on this approach, some authors concluded that the apical glycoproteins in the basolateral membrane fraction were transiently integrated before the apical markers reached the BB domain (Hauri et al, 1979;Quaroni et al, 1979). Others postulated that an intracellular sorting of plasma membrane glycoproteins was involved and that the apical markers used took a direct pathway from the Golgi apparatus to the BB domain (Ahnen et al, 1982;Danielsen and Cowell, 1985). The discrepancies between the results of all these studies have always been blamed on the subcellular fractionation procedures used, the purity of the membrane fractions, and the possible cross-contamination of the basolateral membrane fraction by the intracellular membrane fraction (e.g., Golgi apparatus, intracellular vesicles).…”