The functional roles of disulfide bonds in the beta-subunit of (Na,K)ATPase as studied by site-directed mutagenesis.
The beta-subunit of Torpedo californica (Na,K)ATPase contains seven cysteine residues; one (Cys46) is in the single transmembrane segment and the other six (Cys127, Cys150, Cys160, Cys176, Cys215 and Cys278) are in the extracellular domain and form three highly conserved disulfide bonds. A beta-subunit mutant with replacement of Cys46 by Ser could assemble with the alpha-subunit, and the resulting alpha beta-complex was catalytically active. Mutants in which either the N-terminal side or both Cys residues of the Cys127-Cys150 bond were replaced by Ser could also tightly assemble with the alpha-subunit, but the resulting alpha beta-complex was catalytically inactive. On the other hand, disruption of either the Cys160-Cys176 or Cys215-Cys278 bond by substituting the N-terminal side only or both Cys residues with Ser led to a beta-subunit that could not assemble with the alpha-subunit. We conclude that the structure of the beta-subunit around the Cys160-Cys176 and Cys215-Cys278 loops is indispensable for assembly with the alpha-subunit, whereas the Cys127-Cys150 loop is not essential for assembly but is required for enzyme activity.
Noguchi
S
S
Department Biology, University of Occupational and Environmental Health, Kitakyushu, Japan.
Mutoh
Y
Y
Kawamura
M
M
D000595:Amino Acid Sequence; D000818:Animals; D004220:Disulfides / Q000378:metabolism*; D006801:Humans; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D012333:RNA, Messenger / Q000235:genetics; D017386:Sequence Homology, Amino Acid; D000254:Sodium-Potassium-Exchanging ATPase / Q000235:genetics / Q000378:metabolism*; D014101:Torpedo; D014981:Xenopus
D004220:Disulfides; D012333:RNA, Messenger; D000254:Sodium-Potassium-Exchanging ATPase