Ankyrin Facilitates Intracellular Trafficking of α1-Na-ATPase in polarized cells

Defects in ankyrin underlie many hereditary disorders involving the mis-localization of membrane proteins. Such phenotypes are usually attributed to ankyrin’s role in stabilizing a plasma membrane scaffold, but this assumption may not be accurate. We find in Madin-Darby canine kidney (MDCK) and in other cultured cells that the 25 residue ankyrin-binding sequence of α1-Na,K-ATPase facilitates the entry of α1β1-Na,K-ATPase into the secretory pathway, and that replacement of the cytoplasmic domain of vesicular stomatitis virus G protein (VSV-G) with this ankyrin-binding sequence bestows ankyrindependency on the ER-to-Golgi trafficking of VSV-G. Expression of the ankyrin-binding sequence of α1-Na,K-ATPase alone as a soluble cytosolic peptide acts in trans to selectively block ER to Golgi transport of wild-type α1-Na,K-ATPase and a VSV-G fusion protein that includes the ankyrin-binding sequence, while the trafficking of other proteins remain unaffected. Similar phenotypes are also generated by shRNA-mediated knockdown of ankyrin-R or the depletion of ankyrin in semi-permeabilized cells. These data indicate that the adapter protein ankyrin acts not only at the plasma membrane, but also early in the secretory pathway to facilitate the intracellular trafficking of α1-Na,KATPase and presumably other selected proteins. This novel ankyrin-dependent assembly pathway suggests a mechanism whereby hereditary disorders of ankyrin may be manifested as diseases of membrane protein ER-retention or mis-localization.