Abstract
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.
Highlights
FXYD domain-containing proteins are tissue-specific regulators of the Na,K-ATPase that have been shown to have significant physiological implications
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 ␣-subunit of Na,K-ATPase, using covalent cross-linking combined with proteolytic cleavage
Summary
FXYD domain-containing proteins are tissue-specific regulators of the Na,K-ATPase that have been shown to have significant physiological implications. The present study was undertaken to identify the location of the C-terminal domain of FXYD10 on the ␣-subunit of Na,K-ATPase, using covalent cross-linking combined with proteolytic cleavage.
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