Structural determinants required for apical sorting of an intestinal brush-border membrane protein.
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(DeltaST) (stalk-free) and pro-SI(DeltaMA) (membrane anchor-free), were constructed and expressed in polarized Madin-Darby canine kidney cells. In the absence of the membrane anchoring domain, pro-SI(DeltaMA) 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(DeltaST) does not associate either with lipid rafts and its targeting behavior is similar to that of pro-SI(DeltaMA). 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.
Jacob
R
R
Department of Physiological Chemistry, School of Veterinary Medicine Hannover, Bünteweg 17, D-30559 Hannover, Germany.
Alfalah
M
M
Grünberg
J
J
Obendorf
M
M
Naim
H Y
HY
D000818:Animals; D001692:Biological Transport / Q000235:genetics; D002460:Cell Line; D002499:Centrifugation, Density Gradient; D004285:Dogs; D006031:Glycosylation; D007421:Intestine, Small / Q000201:enzymology / Q000378:metabolism*; D007700:Kinetics; D050356:Lipid Metabolism; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics*; D008871:Microvilli / Q000378:metabolism*; D017384:Sequence Deletion; D013394:Sucrase-Isomaltase Complex / Q000737:chemistry / Q000235:genetics*; D014162:Transfection
D008565:Membrane Proteins; D013394:Sucrase-Isomaltase Complex