The functional roles of disulfide bonds in the β-subunit of (Na,K)ATPase as studied by site-directed mutagenesis

Abstract

The β-subunit of Torpedo californica (Na,K)ATPase contains seven cysteine residues; one (Cys 46) is in the single transmembrane segment and the other six (Cys 127, Cys 150, Cys 160, Cys 176, Cys 215 and Cys 278) are in the extracellular domain and form three highly conserved disulfide bonds. Aβ-subunit mutant with replacement of Cys 46 by Ser could assemble with the α-subunit, and the resulting αβ-complex was catalytically active. Mutants in which either the N-terminal side or both Cys residues of the Cys 127-Cys 150 bond were replaced by Ser could also tightly assemble with the α-subunit, but the resulting αβ-complex was catalytically inactive. On the other hand, disruption of either the Cys 160-Cys 176 or Cys 215-Cys 278 bond by substituting the N-terminal side only or both Cys residues with Ser led to a β-subunit that could not assemble with the α-subunit. We conclude that the structure of the β-subunit around the Cys 160-Cys 176 and Cys 215-Cys 278 loops is indispensable for assembly with the α-subunit, whereas the Cys 127-Cys 150 loop is not essential for assembly but is required for enzyme activity.