Structural interactions between FXYD proteins and Na+,K+-ATPase: alpha/beta/FXYD subunit stoichiometry and cross-linking.
Interactions of rat FXYD4 (corticosteroid hormone-induced factor (CHIF)), FXYD2 (gamma), or FXYD1 (phospholemman (PLM)) proteins with rat alpha1 subunits of Na(+),K(+)-ATPase have been analyzed by co-immunoprecipitation and covalent cross-linking. In detergent-solubilized membranes from HeLa cells expressing both gamma and CHIF or CHIF and hemagglutinin A-tagged CHIF, mixed complexes of CHIF and gamma or CHIF and hemagglutinin A-tagged CHIF with alpha/beta subunits are undetectable. This implies that the alpha/beta/FXYD protomer is the major species in detergent solution. A lipid-soluble cysteine-cysteine bifunctional reagent, dibromobimane, cross-links CHIF to alpha in colonic membranes but not gamma or PLM to alpha in kidney or heart membranes, respectively. Sequence comparisons of the FXYD proteins suggested that Cys-49 in the trans-membrane segment of CHIF could be involved. In detergent-solubilized HeLa cell membranes, dibromobimane cross-links wild-type CHIF to alpha but not the C49F mutant, and also the corresponding F36C mutant but not wild-type gammab, and F48C but not wild-type PLM. C140S, C338A, C804A, and C966S mutants of the alpha subunit have been expressed. Only the C140S mutant prevents cross-linking with CHIF. The data demonstrated the proximity of trans-membrane segments of CHIF, gamma, and PLM to M2 of alpha. Molecular modeling is consistent with location of the trans-membrane segment of all FXYD proteins between M2, M6, and M9 and the proximity of Cys-49 of CHIF or Phe-36 of gamma with Cys-140 of M2. Cross-linking also demonstrated CHIF-alpha and CHIF-beta proximities in extra-membrane regions, similar to the evidence for gamma-alpha and gamma-beta cross-links.
Lindzen
Moshit
M
Department of Biological Chemistry, Weizmann Institute of Science, Rehovot 76100, Israel.
Gottschalk
Kay-Eberhard
KE
Füzesi
Maria
M
Garty
Haim
H
Karlish
Steven J D
SJ
D000595:Amino Acid Sequence; D000818:Animals; D002462:Cell Membrane / Q000378:metabolism; D003432:Cross-Linking Reagents / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010750:Phosphoproteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D015221:Potassium Channels / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011487:Protein Conformation; D021122:Protein Subunits / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D051381:Rats; D016415:Sequence Alignment; D000254:Sodium-Potassium-Exchanging ATPase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D003432:Cross-Linking Reagents; C094337:Fxyd4 protein, rat; D047908:Intracellular Signaling Peptides and Proteins; D008565:Membrane Proteins; D010750:Phosphoproteins; D015221:Potassium Channels; D021122:Protein Subunits; C069141:phospholemman; C498817:Fxyd2 protein, rat; D000254:Sodium-Potassium-Exchanging ATPase