Assembly of the (Na+ + K+)-adenosine triphosphatase. Post-translational membrane integration of the alpha subunit.
Guinea pig kidney poly(A+) RNA was translated in reticulocyte lysates and wheat germ extracts. Antibodies to the holoenzyme (Na/K-ATPase) immunoprecipitated only a 96,000-dalton product which was identified as the alpha subunit with a molecular weight that was indistinguishable from that of mature alpha subunit. To explore the possibility that the primary translational product is integrated as such into membranes, guinea pig kidney poly(A+) RNA was translated in reticulocyte lysates in the presence of dog pancreas microsomes; two immunoprecipitated products were detected, the 96,000-dalton alpha subunit and a 135,000-dalton new component that was integrated into the microsomal membrane since it was completely resistant to extraction with alkali. Addition of purified alpha subunit inhibited the binding of antibody to the 135,000-dalton product and extraction with urea-sodium dodecyl sulfate recovered the 96,000-dalton product, implying that the 135,000-dalton product was an alpha-chi dimer. Translation of size-fractionated poly(A+) RNA yielded evidence that the 135,000-dalton product is encoded in two separate mRNAs. The integration in vitro of the alpha subunit is, therefore, dependent on the co-translational integration into the membranes of a smaller peptide (35,000 to 40,000 daltons) which is presumably the beta subunit. Evidence was also obtained that this mechanism is present in vivo by isolation of mRNA alpha from free polysomes, as well as detection of the cytosolic form of the alpha subunit in pulse-chase experiments in MDCK cells.
D000818:Animals; D002462:Cell Membrane / Q000201:enzymology; D006168:Guinea Pigs; D007668:Kidney / Q000201:enzymology*; D046911:Macromolecular Substances; D051379:Mice; D008970:Molecular Weight; D010944:Plants / Q000378:metabolism; D011061:Poly A / Q000235:genetics; D014176:Protein Biosynthesis; D011499:Protein Processing, Post-Translational; D012313:RNA / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics; D012156:Reticulocytes / Q000378:metabolism; D000254:Sodium-Potassium-Exchanging ATPase / Q000235:genetics*; D014908:Triticum / Q000378:metabolism
D046911:Macromolecular Substances; D012333:RNA, Messenger; D011061:Poly A; D012313:RNA; D000254:Sodium-Potassium-Exchanging ATPase