Delivery of newly synthesized Na+-K+-ATPase to the plasma membrane of A6 epithelia

Na+-K+-ATPase is localized to the basolateral cell surface of most epithelial cells. Conflicting results regarding the intracellular trafficking of Na+-K+- ATPase in Madin-Darby canine kidney cells have been reported, with delivery to both apical and basolateral membranes or exclusively to the basolateral cell surface. We examined the delivery and steady-state distribution of Na+-K+-ATPase in the amphibian epithelial cell line A6 using an antibody raised against Na+-K+-ATPase alpha-subunit and sulfo-N-hydroxysuccinimidobiotin to tag cell surface proteins. The steady-state distribution of the Na+-K+-ATPase was basolateral, as confirmed by immunocytochemistry. Delivery of newly synthesized Na+-K+-ATPase to the cell surface was examined using [S-35]methionine and [S-35]cysteine in a pulse-chase protocol. After a 20-min pulse, the alpha-subunit and core glycosylated beta-subunit were present at both apical and basolateral cell surfaces. The alpha-subunit and core glycosylated beta-subunit delivered to the apical cell surface were degraded within 2 h. Mature alpha/beta-heterodimer was found almost exclusively at the basolateral surface after a 1- to 24-h chase. These data suggest that immature Na+-K+-ATPase alpha-subunit and core glycosylated beta-subunits are not retained in the endoplasmic reticulum of A6 cells and apparently lack sorting signals. Mature Na+-K+-ATPase is targeted to the basolateral surface, suggesting that basolateral targeting of the protein is conformation dependent.