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

doi:10.1074/jbc.275.9.6566

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

From the Department of Physiological Chemistry, School of Veterinary Medicine Hannover, Bünteweg 17, D-30559 Hannover, Germany

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_ΔST (stalk-free) and pro-SI_ΔMA (membrane anchor-free), were constructed and expressed in polarized Madin-Darby canine kidney cells. In the absence of the membrane anchoring domain, pro-SI_ΔMA 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_ΔST does not associate either with lipid rafts and its targeting behavior is similar to that of pro-SI_ΔMA. 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 (Ala³⁷-Pro⁴⁸) juxtapose the membrane anchor is required in conjunction with the membrane anchoring domain for correct targeting of pro-SI to the apical membrane. OtherO-glycosylated domains within the stalk (Ala⁴⁹-Pro⁵⁷) are not sufficient for apical sorting. We conclude that the recognition signal for apical sorting of pro-SI comprises O-glycosylation of the Ala³⁷-Pro⁴⁸ stretch and requires the presence of the membrane anchoring domain.

Footnotes

↵* This work was supported by Deutsche Forschungsgemeinschaft Grant Na 331/1-2, Bonn/Germany (to H. Y. Naim), and Sonderforschungsbereich Grant 280.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵‡ To whom correspondence should be addressed: Dept. of Physiological Chemistry, School of Veterinary Medicine, Hannover, Bünteweg 17, D-30559 Hannover, Germany. Tel.: 49-511-953-8780; Fax: 49-511-953-8585; E-mail: hnaim@biochemie.tiho-hannover.de.
Abbreviations:

CHAPS

3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid

SI

sucrase isomaltase (all forms)

pro-SI

uncleaved sucrase isomaltase

mAb

monoclonal antibody

benzyl-GalNAc

benzyl-N-acetyl-α-d-galactosaminide

Endo H

endoglyosidase H

Endo F/GF

endoglycosidase F/N-glycopeptidase F

LPH

lactase-phlorizin hydrolase

MDCK

Madin-Darby canine kidney cells

TEMED

N,N,N′,N′-tetramethylethylenediamine

PAGE

polyacrylamide gel electrophoresis
- Received September 1, 1999.
- Revision received November 30, 1999.
The American Society for Biochemistry and Molecular Biology, Inc.