HIERARCHY OF MECHANISMS INVOLVED IN GENERATING NA K-ATPASE POLARITY IN MDCK EPITHELIAL-CELLS/

We have studied mechanisms involved in generating a polarized distribu tion of Na/K-ATPase in the basal-lateral membrane of two clones of MDC K II cells. Both clones exhibit: polarized distributions of marker pro teins of the apical and basal-lateral membranes, including Na/K-ATPase , at steady state, Newly synthesized Na/K-ATPase, however, is delivere d from the Golgi complex to both apical and basal-lateral membranes of one clone (II/J), and to the basal-lateral membrane of the other clon e (II/G); Na/K-ATPase is selectively retained in the basal-lateral mem brane resulting in the generation of complete cell surface polarity in both clones. Another basal-lateral membrane protein, E-cadherin, is s orted to the basal-lateral membrane in both MDCK clones, demonstrating that there is not a general sorting defect for basal-lateral membrane proteins in clone II/J cells, A glycosyl-phosphatidylinositol (GPI-)- anchored protein (GP-2) and a glycosphingolipid (glucosylceramide, Glc Cer) are preferentially transported to the apical membrane in clone II /G cells, but, in clone II/J cells, GP-2 and GlcCer are delivered equa lly to both apical and basal-lateral membranes, similar to Na/K-ATPase . To examine this apparent inter-relationship between sorting of GlcCe r, GP-2 and Na/K-ATPase, sphingolipid synthesis was inhibited in clone II/G cells with the fungal metabolite, Fumonisin B-1 (FB1), In the pr esence of FB1, GP-2 and Na/K-ATPase are delivered to both apical and b asal-lateral membranes, similar to clone II/J cells; FB1 had no effect on sorting of E-cadherin to the basal-lateral membrane of II/G cells. Addition of exogenous ceramide, to circumvent the FB1 block, restored GP-2 and Na/K-ATPase sorting to the apical and basal-lateral membrane s, respectively. These results show that the generation of complete ce ll surface polarity of Na/K-ATPase involves a hierarchy of sorting mec hanisms in the Golgi complex and plasma membrane, and that Na/K-ATPase sorting in the Golgi complex of MDCK cells may be regulated by exclus ion from an apical pathway(s). These results also provide new insights into sorting pathways for other apical and basal-lateral membrane pro teins.