Mutational analysis of alpha-beta subunit interactions in the delivery of Na,K-ATPase heterodimers to the plasma membrane.
The beta-subunit of the Na,K-ATPase is required to deliver functional alpha beta-heterodimers to the plasma membrane (PM) of baculovirus-infected insect cells. We have investigated the molecular determinants in the beta-subunit for the assembly and delivery processes. Trafficking of both subunits was analyzed by Western blots of fractionated membranes enriched in endoplasmic reticulum (ER), Golgi, and PM. Heterodimer assembly was evaluated by co-immunoprecipitation, and enzymatic activity was measured by ATPase assay. Elimination of enzymatic activity by D369A point mutation of the alpha-subunit had no effect on the compartmental distribution of the Na,K-ATPase, demonstrating that enzymatic functioning is not a prerequisite for PM delivery. Replacement of all three N-glycosylation site asparagines with glutamines produced no effect on the delivery to the PM or the activity of the enzyme, but increased susceptibility to degradation was observed. Analysis of beta-subunits in which the disulfide bonds were removed through substitution reveals that the bridges are important for PM targeting but not for assembly of the heterodimer. Assembly is supported by beta-subunits with greatly truncated extracellular domains. The presence of the amino-terminal domain and transmembrane segment is sufficient for assembly and PM delivery. Intermediate length truncated beta-subunits and some disulfide bridge substitution mutants assemble with the alpha-subunit but are not able to exit the ER. We conclude that there are different and separable requirements for the assembly of Na,K-ATPase heterodimer complexes, exit of the dimer from the ER, delivery to the PM, and catalytic activity of the dimer.
Laughery
Melissa D
MD
Department of Biochemistry and Molecular Biology, Oregon Health and Sciences University, Portland, Oregon 97239, USA.
Todd
Matthew L
ML
Kaplan
Jack H
JH
D019943:Amino Acid Substitution; D000818:Animals; D002460:Cell Line; D002462:Cell Membrane / Q000201:enzymology / Q000378:metabolism; D019281:Dimerization; D004721:Endoplasmic Reticulum / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D021122:Protein Subunits; D021381:Protein Transport; D011994:Recombinant Proteins / Q000378:metabolism; D012756:Sheep; D000254:Sodium-Potassium-Exchanging ATPase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D018411:Spodoptera; D014162:Transfection
D021122:Protein Subunits; D011994:Recombinant Proteins; D000254:Sodium-Potassium-Exchanging ATPase