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 beta 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 beta 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 beta subunits associate with the endogenous Na,K-ATPase alpha(1) subunits in MDCK but not in LLC-PK cells. Hence, co-sorting of the H,K-ATPase beta subunit with the Na,K-ATPase alpha(1) subunit to the basolateral membrane in MDCK cells may determine the differential distribution of the beta subunit in these two cell types. The major fraction of unassociated monomeric H,K-ATPase beta subunits is detected in the apical membrane. Quantitative analysis showed that half of the apical pool of the beta 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 beta 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 beta subunit in 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.
Highlights
Ered directly either to the apical or basolateral membranes [1,2,3,4,5]
The results of these studies indicate that membrane proteins containing both apical and basolateral sorting signals, such as the gastric H,K-ATPase  subunit, which are resident in the apical membrane, can arrive there by both direct delivery from the TGN and transcytosis via the basolateral membrane
Steady State Surface Distribution of the Wild Type and Mutant YFP- in Polarized Madin-Darby canine kidney (MDCK) and LLC-PK1 Cells—Expression of the gastric H,K-ATPase  subunit as a YFP N-terminal construct in eukaryotic cells results in a synthesis of a fusion protein that retains correct folding, post-translational modifications, and ability to assemble with its ␣ subunit and supports the active conformation of the ␣, complex, as was shown before in nonpolarized HEK-293 cells [24] and polarized LLCPK1 cells [18]
Summary
Vol 280, No 15, Issue of April 15, pp. 14741–14754, 2005 Printed in U.S.A. Use of the H,K-ATPase  Subunit to Identify Multiple Sorting Pathways for Plasma Membrane Delivery in Polarized Cells*. The gastric H,K-ATPase  subunit has several properties that make it an excellent model to use in studies of the sorting pathways involved in distribution of membrane proteins and the factors modulating the sorting process It can be expressed alone or in association with its natural partner or other subunits in cultured cells, it has embedded in it both apical and basolateral sorting signals, and it is delivered predominantly to the apical or basolateral membranes in different polarized cell systems. We found that promiscuous association with the endogenous Na,K-ATPase ␣1 subunit in MDCK but not LLCPK1 cells is probably responsible for differential distribution of the H,K-ATPase  subunit in the two cell types The results of these studies indicate that membrane proteins containing both apical and basolateral sorting signals, such as the gastric H,K-ATPase  subunit, which are resident in the apical membrane, can arrive there by both direct delivery from the TGN and transcytosis via the basolateral membrane. These data emphasize that sorting is a complex process involving an interaction between sorting machinery unique to specific cells, signals embedded in proteins, and, in the case of heterodimeric proteins, the relative dominance of the sorting information in the particular subunit
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