Interaction of FXYD10 (PLMS) with Na,K-ATPase from shark rectal glands. Close proximity of Cys74 of FXYD10 to Cys254 in the a domain of the alpha-subunit revealed by intermolecular thiol cross-linking.
FXYD domain-containing proteins are tissue-specific regulators of the Na,K-ATPase that have been shown to have significant physiological implications. Information about the sites of interaction between some FXYD proteins and subunits of the Na,K-ATPase is beginning to emerge. We previously identified an FXYD protein in plasma membranes from shark rectal gland cells and demonstrated that this protein (FXYD10) modulates shark Na,K-ATPase activity. The present study was undertaken to identify the location of the C-terminal domain of FXYD10 on the alpha-subunit of Na,K-ATPase, using covalent cross-linking combined with proteolytic cleavage. Treatment of Na,K-ATPase-enriched membranes with the homobifunctional thiol cross-linker 1,4-bismaleimidyl-2,3-dihydroxybutane resulted in cross-linking of FXYD10 to the alpha-subunit. Cross-linking was not affected by preincubation with sodium or potassium but was significantly reduced after pre-incubation with the non-hydrolyzable ATP analog beta,gamma-methyleneadenosine 5'-triphosphate (AMP-PCP). A peptic assay was developed, in which pepsin treatment of Na,K-ATPase at low pH resulted in extensive cleavage of the alpha-subunit while FXYD10 was left intact. Proteolytic fragments of control and cross-linked preparations were isolated by immunoprecipitation and analyzed by gel electrophoresis. A proteolytic fragment containing FXYD10 cross-linked to a fragment from the alpha-subunit could be localized on SDS gels. Sequencing of this fragment showed the presence of FXYD10 as well as a fragment within the A domain of the alpha-subunit comprising 33 amino acids, including a single Cys residue, Cys254. Thus, regulation of Na,K-ATPase by FXYD10 occurs in part via cytoplasmic interaction of FXYD10 with the A domain of the shark alpha-subunit.
Mahmmoud
Yasser Ahmed
YA
Institute of Physiology and Biophysics, University of Aarhus, DK-8000 Aarhus C, Denmark. yam@biophys.au.dk
Vorum
Henrik
H
Cornelius
Flemming
F
D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000737:chemistry / Q000494:pharmacology; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002072:Butylene Glycols / Q000494:pharmacology; D002462:Cell Membrane / Q000378:metabolism; D018118:Chloride Channels; D003432:Cross-Linking Reagents / Q000494:pharmacology; D003545:Cysteine / Q000737:chemistry; D003593:Cytoplasm / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D029941:Fish Proteins / Q000737:chemistry / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D015151:Immunoblotting; D047468:Immunoprecipitation; D008024:Ligands; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010434:Pepsin A / Q000737:chemistry / Q000494:pharmacology; D010750:Phosphoproteins / Q000737:chemistry / Q000378:metabolism*; D010766:Phosphorylation; D011188:Potassium / Q000737:chemistry; D011485:Protein Binding; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D012964:Sodium / Q000737:chemistry; D000254:Sodium-Potassium-Exchanging ATPase / Q000737:chemistry / Q000378:metabolism*; D048251:Squalus acanthias; D013438:Sulfhydryl Compounds / Q000737:chemistry; D014451:Ubiquinone / Q000031:analogs & derivatives / Q000494:pharmacology
D002072:Butylene Glycols; D018118:Chloride Channels; D003432:Cross-Linking Reagents; D029941:Fish Proteins; D008024:Ligands; C417182:PLMS protein, Squalus acanthias; D010750:Phosphoproteins; D013438:Sulfhydryl Compounds; D014451:Ubiquinone; C005147:5'-adenylyl (beta,gamma-methylene)diphosphonate; C060262:2,3-dimethoxy-5-methyl-6-decyl-1,4-benzoquinone; D000255:Adenosine Triphosphate; D012964:Sodium; D010434:Pepsin A; D000254:Sodium-Potassium-Exchanging ATPase; D003545:Cysteine; D011188:Potassium