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 [35S]methionine and [35S]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.
Coupaye-Gerard
B
B
Department of Medicine, University of Pennsylvania, Philadelphia, USA.
Zuckerman
J B
JB
Duncan
P
P
Bortnik
A
A
Avery
D I
DI
Ernst
S A
SA
Kleyman
T R
TR
D000818:Animals; D002460:Cell Line; D002462:Cell Membrane / Q000201:enzymology* / Q000648:ultrastructure; D004285:Dogs; D004847:Epithelial Cells; D004848:Epithelium / Q000201:enzymology; D007668:Kidney / Q000201:enzymology; D008954:Models, Biological; D011499:Protein Processing, Post-Translational; D000254:Sodium-Potassium-Exchanging ATPase / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D014982:Xenopus laevis
D000254:Sodium-Potassium-Exchanging ATPase