Use of the H,K-ATPase beta subunit to identify multiple sorting pathways for plasma membrane delivery in polarized cells.
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.
Vagin
Olga
O
Department of Physiology, School of Medicine, UCLA and Veterans Administration Greater Los Angeles Health Care System, Los Angeles, California 90073, USA. olgav@ucla.edu <olgav@ucla.edu>
Turdikulova
Shahlo
S
Yakubov
Iskandar
I
Sachs
George
G
D000818:Animals; D001426:Bacterial Proteins / Q000378:metabolism; D001692:Biological Transport; D001710:Biotin / Q000737:chemistry; D019921:Biotinylation; D015153:Blotting, Western; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism*; D003513:Cycloheximide / Q000494:pharmacology; D018076:DNA, Complementary / Q000378:metabolism; D004285:Dogs; D004591:Electrophoresis, Polyacrylamide Gel; D004705:Endocytosis; D011992:Endosomes / Q000378:metabolism; D006056:Golgi Apparatus / Q000378:metabolism; D017506:H(+)-K(+)-Exchanging ATPase / Q000737:chemistry*; D008164:Luminescent Proteins / Q000378:metabolism; D018613:Microscopy, Confocal; D009154:Mutation; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D015398:Signal Transduction; D013552:Swine; D013696:Temperature; D013997:Time Factors; D014162:Transfection; D014443:Tyrosine / Q000737:chemistry
D001426:Bacterial Proteins; D018076:DNA, Complementary; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; C054562:yellow fluorescent protein, Bacteria; D014443:Tyrosine; D001710:Biotin; D003513:Cycloheximide; D017506:H(+)-K(+)-Exchanging ATPase