Structural determinants required for apical sorting of an intestinal brush-border membrane protein.

Area of Science:

• Cell biology
• Molecular biology
• Biochemistry

Background:

• Polarized cells exhibit distinct apical and basolateral membrane protein and lipid compositions.
• O-Glycosylation of sucrase isomaltase (SI) is linked to its apical sorting via sphingolipid-cholesterol microdomains.

Purpose of the Study:

• To identify the structural determinants governing the apical sorting of the intestinal protein sucrase isomaltase (SI).
• To investigate the roles of the membrane anchor and the Ser/Thr-rich stalk domain in SI's polarized delivery.

Main Methods:

• Construction and expression of deletion mutants of pro-SI (pro-SI(DeltaST) and pro-SI(DeltaMA)) in polarized Madin-Darby canine kidney cells.
• Analysis of mutant protein association with lipid rafts and their localization to cellular membranes.

Main Results:

• The membrane anchoring domain is crucial for lipid raft association and apical targeting; its absence leads to random membrane distribution.
• The O-glycosylated stalk region alone is insufficient for high-fidelity apical sorting.
• Only wild-type pro-SI, possessing both the stalk and membrane anchor, correctly associates with lipid microdomains and targets the apical membrane.
• A specific 12-amino acid O-glycosylated stretch (Ala(37)-Pro(48)) adjacent to the membrane anchor is required for apical sorting.

Conclusions:

• Apical sorting of pro-SI requires the concerted action of its membrane anchoring domain and O-glycosylation of the Ala(37)-Pro(48) stretch.
• The identified O-glycosylation site and membrane anchor form a recognition signal for apical membrane delivery.