Oligomerization of the Na,K-ATPase in cell membranes.
The higher order oligomeric state of the Na,K-ATPase alphabeta heterodimer in cell membranes is the subject of controversy. We have utilized the baculovirus-infected insect cell system to express Na,K-ATPase with alpha-subunits bearing either His(6) or FLAG epitopes at the carboxyl terminus. Each of these constructs produced functional Na,K-ATPase alphabeta heterodimers that were delivered to the plasma membrane (PM). Cells were simultaneously co-infected with viruses encoding alpha-His/beta and alpha-FLAG/beta Na,K-ATPases. Co-immunoprecipitation of the His-tagged alpha-subunit in the endoplasmic reticulum (ER) and PM fractions of co-infected cells by the anti-FLAG antibody demonstrates that protein-protein associations exist between these heterodimers. This suggests the Na,K-ATPase is present in cell membranes in an oligomeric state of at least (alphabeta)(2) composition. Deletion of 256 amino acid residues from the central cytoplasmic loop of the alpha-subunit results in the deletion alpha-4,5-loop-less (alpha-4,5LL), which associates with beta but is confined to the ER. Co-immunoprecipitation demonstrates that when this inactive alpha-4,5LL/beta heterodimer is co-expressed with wild-type alphabeta, oligomers of wild-type alphabeta and alpha-4,5LL/beta form in the ER, but the alpha-4,5LL mutant remains retained in the ER, and the wild-type protein is still delivered to the PM. We conclude that the Na,K-ATPase is present as oligomers of the monomeric alphabeta heterodimer in native cell membranes.
Laughery
Melissa
M
Department of Biochemistry and Molecular Genetics, University of Illinois at Chicago, 900 S. Ashland Avenue, Chicago, IL 60607-7170, USA.
Todd
Matthew
M
Kaplan
Jack H
JH
D000818:Animals; D016367:Baculoviridae / Q000378:metabolism; D015153:Blotting, Western; D002460:Cell Line; D002462:Cell Membrane / Q000201:enzymology* / Q000378:metabolism; D003593:Cytoplasm / Q000378:metabolism; D018076:DNA, Complementary / Q000378:metabolism; D019281:Dimerization; D004721:Endoplasmic Reticulum / Q000378:metabolism; D000939:Epitopes / Q000737:chemistry; D017353:Gene Deletion; D007313:Insecta; D009154:Mutation; D010766:Phosphorylation; D010957:Plasmids / Q000378:metabolism; D011233:Precipitin Tests; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D000254:Sodium-Potassium-Exchanging ATPase / Q000737:chemistry* / Q000378:metabolism; D014357:Trypsin / Q000494:pharmacology
D018076:DNA, Complementary; D000939:Epitopes; D014357:Trypsin; D000254:Sodium-Potassium-Exchanging ATPase