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 epi thelial cells. Conflicting results regarding the intracellular traffic king of Na+-K+- ATPase in Madin-Darby canine kidney cells have been re ported, with delivery to both apical and basolateral membranes or excl usively 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-sub unit 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 a lpha-subunit and core glycosylated beta-subunit were present at both a pical and basolateral cell surfaces. The alpha-subunit and core glycos ylated beta-subunit delivered to the apical cell surface were degraded within 2 h. Mature alpha/beta-heterodimer was found almost exclusivel y at the basolateral surface after a 1- to 24-h chase. These data sugg est that immature Na+-K+-ATPase alpha-subunit and core glycosylated be ta-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 t he protein is conformation dependent.