Neutralization of endomembrane compartments in epithelial MDCK cells affects proteoglycan synthesis in the apical secretory pathway.
PGs (proteoglycans) are proteins acquiring long, linear and sulfated GAG (glycosaminoglycan) chains during Golgi passage. In MDCK cells (Madin-Darby canine kidney cells), most of the CS (chondroitin sulfate) PGs are secreted apically, whereas most of the HS (heparan sulfate) PGs are secreted basolaterally. The apical and basolateral secretory routes differ in their GAG synthesis, since a protein core that traverses both routes acquires shorter chains, but more sulfate, in the basolateral pathway than in the apical counterpart [Tveit, Dick, Skibeli and Prydz (2005) J. Biol. Chem. 280, 29596-29603]. Golgi cisternae and the trans-Golgi network have slightly acidic lumens. We therefore investigated how neutralization of endomembrane compartments with the vacuolar H(+)-ATPase inhibitor Baf A(1) (bafilomycin A(1)) affected GAG synthesis and PG sorting in MDCK cells. Baf A(1) induced a slight reduction in basolateral secretion of macromolecules, which was compensated by an apical increase. More dramatic changes occurred to PG synthesis in the apical pathway on neutralization. The difference in apical and basolateral PG sulfation levels observed for control cells was abolished, due to enhanced sulfation of apical CS-GAGs. In addition, a large fraction of apical HS-GAGs was elongated to longer chain lengths. The differential sensitivity of the apical and basolateral secretory pathways to Baf A(1) indicates that the apical pathway is more acidic than the basolateral counterpart in untreated MDCK cells. Neutralization gave an apical GAG output that was more similar to that of the basolateral pathway, suggesting that neutralization made the luminal environments of the two pathways more similar.
Grøndahl
Frøy
F
Department of Molecular Biosciences, University of Oslo, Blindern, Oslo, Norway.
Tveit
Heidi
H
Prydz
Kristian
K
D000818:Animals; D002478:Cells, Cultured; D002809:Chondroitin Sulfates / Q000096:biosynthesis; D004285:Dogs; D004847:Epithelial Cells / Q000378:metabolism / Q000648:ultrastructure*; D006025:Glycosaminoglycans / Q000096:biosynthesis; D007668:Kidney; D018942:Macrolides / Q000494:pharmacology; D011509:Proteoglycans / Q000096:biosynthesis* / Q000378:metabolism; D055571:Secretory Pathway / Q000502:physiology*; D013463:Sulfuric Acid Esters / Q000378:metabolism; D025262:Vacuolar Proton-Translocating ATPases / Q000037:antagonists & inhibitors* / Q000235:genetics
D006025:Glycosaminoglycans; D018942:Macrolides; D011509:Proteoglycans; D013463:Sulfuric Acid Esters; C040929:bafilomycin A1; D002809:Chondroitin Sulfates; D025262:Vacuolar Proton-Translocating ATPases