title: FXYD proteins stabilize Na,K-ATPase: amplification of specific phosphatidylserine-protein interactions.
abstract: FXYD proteins are a family of seven small regulatory proteins, expressed in a tissue-specific manner, that associate with Na,K-ATPase as subsidiary subunits and modulate kinetic properties. This study describes an additional property of FXYD proteins as stabilizers of Na,K-ATPase. FXYD1 (phospholemman), FXYD2 (γ subunit), and FXYD4 (CHIF) have been expressed in Escherichia coli and purified. These FXYD proteins associate spontaneously in vitro with detergent-soluble purified recombinant human Na,K-ATPase (α1β1) to form α1β1FXYD complexes. Compared with the control (α1β1), all three FXYD proteins strongly protect Na,K-ATPase activity against inactivation by heating or excess detergent (C(12)E(8)), with effectiveness FXYD1 > FXYD2 ≥ FXYD4. Heating also inactivates E(1) ↔ E(2) conformational changes and cation occlusion, and FXYD1 protects strongly. Incubation of α1β1 or α1β1FXYD complexes with guanidinium chloride (up to 6 m) causes protein unfolding, detected by changes in protein fluorescence, but FXYD proteins do not protect. Thus, general protein denaturation is not the cause of thermally mediated or detergent-mediated inactivation. By contrast, the experiments show that displacement of specifically bound phosphatidylserine is the primary cause of thermally mediated or detergent-mediated inactivation, and FXYD proteins stabilize phosphatidylserine-Na,K-ATPase interactions. Phosphatidylserine probably binds near trans-membrane segments M9 of the α subunit and the FXYD protein, which are in proximity. FXYD1, FXYD2, and FXYD4 co-expressed in HeLa cells with rat α1 protect strongly against thermal inactivation. Stabilization of Na,K-ATPase by three FXYD proteins in a mammalian cell membrane, as well the purified recombinant Na,K-ATPase, suggests that stabilization is a general property of FXYD proteins, consistent with a significant biological function.
authors.affiliation: Department of Biological Chemistry, Weizmann Institute of Science, Rehovoth 76100, Israel.
authors.forename: Neeraj Kumar
authors.initials: NK
authors.lastname: Mishra
authors.forename: Yoav
authors.initials: Y
authors.lastname: Peleg
authors.forename: Erica
authors.initials: E
authors.lastname: Cirri
authors.forename: Talya
authors.initials: T
authors.lastname: Belogus
authors.forename: Yael
authors.initials: Y
authors.lastname: Lifshitz
authors.forename: Dennis R
authors.initials: DR
authors.lastname: Voelker
authors.forename: Hans-Juergen
authors.initials: HJ
authors.lastname: Apell
authors.forename: Haim
authors.initials: H
authors.lastname: Garty
authors.forename: Steven J D
authors.initials: SJ
authors.lastname: Karlish
mesh_terms: D000818:Animals; D006367:HeLa Cells; D006358:Hot Temperature; D006801:Humans; D008565:Membrane Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D046912:Multiprotein Complexes / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D010718:Phosphatidylserines / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D010750:Phosphoproteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D017510:Protein Folding; D051381:Rats; D000254:Sodium-Potassium-Exchanging ATPase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
chemical_list: D008565:Membrane Proteins; D046912:Multiprotein Complexes; D010718:Phosphatidylserines; D010750:Phosphoproteins; D000254:Sodium-Potassium-Exchanging ATPase