Mutational study on the roles of disulfide bonds in the beta-subunit of gastric H+,K+-ATPase.
The gastric proton pump, H(+),K(+)-ATPase, consists of the catalytic alpha-subunit and the non-catalytic beta-subunit. Correct assembly between the alpha- and beta-subunits is essential for the functional expression of H(+),K(+)-ATPase. The beta-subunit contains nine conserved cysteine residues; two are in the cytoplasmic domain, one in the transmembrane domain, and six in the ectodomain. The six cysteine residues in the ectodomain form three disulfide bonds. In this study, we replaced each of the cysteine residues of the beta-subunit with serine individually and in several combinations. The mutant beta-subunits were co-expressed with the alpha-subunit in human embryonic kidney 293 cells, and the role of each cysteine residue or disulfide bond in the alpha/beta assembly, stability, and cell surface delivery of the alpha- and beta-subunits and H(+),K(+)-ATPase activity was studied. Mutant beta-subunits with a replacement of the cytoplasmic and transmembrane cysteines preserved H(+),K(+)-ATPase activity. All the mutant beta-subunits with replacement(s) of the extracellular cysteines did not assemble with the alpha-subunit, resulting in loss of H(+),K(+)-ATPase activity. These mutants did not permit delivery of the alpha-subunit to the cell surface. Therefore, each of these disulfide bonds of the beta-subunit is essential for assembly with the alpha-subunit and expression of H(+),K(+)-ATPase activity as well as for cell surface delivery of the alpha-subunit.
Kimura
Tohru
T
Faculty of Pharmaceutical Sciences and Molecular Genetics Research Center of Toyama Medical and Pharmaceutical University, 2630 Sugitani, Toyama 930-0194, Japan.
Tabuchi
Yoshiaki
Y
Takeguchi
Noriaki
N
Asano
Shinji
S
D002460:Cell Line; D003545:Cysteine / Q000378:metabolism; D018076:DNA, Complementary; D004220:Disulfides / Q000378:metabolism*; D004795:Enzyme Stability; D017506:H(+)-K(+)-Exchanging ATPase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D007150:Immunohistochemistry; D016297:Mutagenesis, Site-Directed; D013270:Stomach / Q000201:enzymology*
D018076:DNA, Complementary; D004220:Disulfides; D017506:H(+)-K(+)-Exchanging ATPase; D003545:Cysteine