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

Brigitte Coupaye-Gerard, Jonathan B. Zuckerman, Pamela Duncan, Anna Bortnik, Daniel I. Avery, Stephen A. Ernst, Thomas R. Kleyman*

*Corresponding author for this work

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Abstract

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 α-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 α-subunit and core glycosylated β-subunit were present at both apical and basolateral cell surfaces. The α-subunit and core glycosylated β-subunit delivered to the apical cell surface were degraded within 2 h. Mature α/β-heterodimer was found almost exclusively at the basolateral surface after a 1- to 24-h chase. These data suggest that immature Na+-K+-ATPase α-subunit and core glycosylated β-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.

Original languageEnglish
Pages (from-to)C1781-C1789
Number of pages9
JournalAmerican Journal of Physiology - Cell Physiology
Volume272
Issue number6 41-6
Publication statusPublished - 1997
Externally publishedYes

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Keywords

  • Biotin
  • Intracellular trafficking
  • Sodium pump
  • Sodium-potassium adenosinetriphosphatase
  • Sorting

Cite this

Coupaye-Gerard, B., Zuckerman, J. B., Duncan, P., Bortnik, A., Avery, D. I., Ernst, S. A., & Kleyman, T. R. (1997). Delivery of newly synthesized Na+-K+-ATPase to the plasma membrane of A6 epithelia. American Journal of Physiology - Cell Physiology, 272(6 41-6), C1781-C1789.