Structural Determinants Required for Apical Sorting of an Intestinal Brush-border Membrane Protein

Abstract

The distinct protein and lipid constituents of the apical and basolateral membranes in polarized cells are sorted by specific signals. O-Glycosylation of a highly polarized intestinal brush-border protein sucrase isomaltase is implicated in its apical sorting through interaction with sphingolipid-cholesterol microdomains. We characterized the structural determinants required for this mechanism by focusing on two major domains in pro-SI, the membrane anchor and the Ser/Thr-rich stalk domain. Deletion mutants lacking either domain, pro-SI ⌬ST (stalk-free) and pro-SI ⌬MA (membrane anchorfree), were constructed and expressed in polarized Madin-Darby canine kidney cells. In the absence of the membrane anchoring domain, pro-SI ⌬MA does not associate with lipid rafts and the mutant is randomly delivered to both membranes. Therefore, the O-glycosylated stalk region is not sufficient per se for the high fidelity of apical sorting of pro-SI. Pro-SI ⌬ST does not associate either with lipid rafts and its targeting behavior is similar to that of pro-SI ⌬MA . Only wild type pro-SI containing both determinants, the stalk region and membrane anchor, associates with lipid microdomains and is targeted correctly to the apical membrane. However, not all sequences in the stalk region are required for apical sorting. Only O-glycosylation of a stretch of 12 amino acids (Ala 37 -Pro 48 ) juxtapose the membrane anchor is required in conjunction with the membrane anchoring domain for correct targeting of pro-SI to the apical membrane. Other O-glycosylated domains within the stalk (Ala 49 -Pro 57 ) are not sufficient for apical sorting. We conclude that the recognition signal for apical sorting of pro-SI comprises O-glycosylation of the Ala 37 -Pro 48 stretch and requires the presence of the membrane anchoring domain.

A Point Mutation in the Juxtamembrane Stalk of Human Angiotensin I-converting Enzyme Invokes the Action of a Distinct Secretase

“…This view is supported by several examples of proteins from which the transmembrane domains have been eliminated. Membranebound intestinal sucrase-isomaltase, for instance, exhibits similar transport kinetics as its soluble anchorless isoform (35). Likewise, deletion of the transmembrane domain in the neurotrophin receptor has no implications on the intracellular transport of the mutant protein (36).…”

“…After immunoprecipitation, the protein A-Sepharose beads were washed three times with washing buffer A (0.5% Triton X-100 and 0.05% sodium deoxycholate in PBS) and three times with washing buffer B (500 mM NaCl, 10 mM EDTA, and 0.5% Triton X-100 in 125 mM Tris/HCl, pH 8.0) prior to analysis of the samples by SDS-polyacrylamide gel electrophoresis and fluorography as described previously (24). The digestion of 35 S-labeled immunoprecipitates with endo-␤-N-acetylglucosaminidase (Endo) H and Endo F/glycopeptidase F was performed as previously described (24).…”

1-Benzyl-2-acetamido-2-deoxy-α-D-galactopyranoside Blocks the Apical Biosynthetic Pathway in Polarized HT-29 Cells

“…Thus, proteins of apical destination, such as GPI-anchored proteins, would be specifically recruited in these apical raft carriers. Besides, data obtained by mutation, deletion, or addition of glycosylation sites showed that Oor N-glycosylation could influence the targeting of glycoproteins toward the apical side of the cells (5)(6)(7)(8)(9)(10)(11)(12)(13)(14).…”

Characteristics and Structural Requirements of Apical Sorting of the Rat Growth Hormone through the O-Glycosylated Stalk Region of Intestinal Sucrase-isomaltase

“…We examined the role of O-glycosylation in the apical sorting event by making chimeras comprising unglycosylated rat GH as a reporter gene and the Ser/Thr-rich stalk domain and the transmembrane domain of human sucrase-isomaltase. In the wild type SI protein the Ser/Thr-stalk domain is heavily O-glycosylated and the transmembrane domain is required for SI to enter into cholesterol/ sphingolipid-rich membrane microdomains (19). Using wild type rat GH in these chimeras rather than a mutagenized form of rat GH in which potential N-or O-glycosylation sites are introduced should provide direct evidence regarding the location of the apical sorting signal in the stalk region.…”

“…O-Glycosylation is also critically important in the sorting event of some membrane glycoproteins (18). Specific inhibition of O-glycosylation of intestinal sucrase-isomaltase with benzyl-GalNAc (benzyl-N-acetyl-␣-D-galactosaminide) as well as deletion of the potentially O-glycosylated Ser/Thr-rich stalk domain abolished the high fidelity of apical sorting of SI and resulted in a random transport of the protein to both membranes (19,20). Likewise, a dominantly O-glycosylated domain juxtapose the membrane of the neurotrophin receptor is presumably implicated in its apical sorting (14,21).…”