Use of the H,K-ATPase β subunit to identify multiple sorting pathways for plasma membrane delivery in polarized cells

Abstract

A dynamic equilibrium between multiple sorting pathways maintains polarized distribution of plasma membrane proteins in epithelia. To identify sorting pathways for plasma membrane delivery of the gastric H,K-ATPase β subunit in polarized cells, the protein was expressed as a yellow fluorescent protein N-terminal construct in Madin-Darby canine kidney (MDCK) and LLC-PK1 cells. Confocal microscopy and surface-selective biotinylation showed that 80% of the surface amount of the β subunit was present on the apical membrane in LLC-PK1 cells, but only 40% was present in MDCK cells. Nondenaturing gel electrophoresis of the isolated membranes showed that a significant fraction of the H,K-ATPase β subunits associate with the endogenous Na,K-ATPase α1 subunits in MDCK but not in LLC-PK cells. Hence, co-sorting of the H,K-ATPase β subunit with the Na,K-ATPase α1 subunit to the basolateral membrane in MDCK cells may determine the differential distribution of the β subunit in these two cell types. The major fraction of unassociated monomeric H,K-ATPase β subunits is detected in the apical membrane. Quantitative analysis showed that half of the apical pool of the β subunit originates directly from the trans-Golgi network and the other half from transcytosis via the basolateral membrane in MDCK cells. A minor fraction of monomeric β subunits detected in the basolateral membrane represents a transient pool of the protein that undergoes transcytosis to the apical membrane. Hence, the steady state distribution of the H,K-ATPase β subunit polarized cells depends on the balance between (a) direct sorting from the trans-Golgi network, (b) secondary associative sorting with a partner protein, and (c) transcytosis.