Valine 904, tyrosine 898, and cysteine 908 in Na,K-ATPase alpha subunits are important for assembly with beta subunits.
A 26-amino acid sequence in an extracellular loop of the Na,K-ATPase alpha subunit between membrane-spanning segments 7 and 8 has been shown to bind to the beta subunit of Na,K-ATPase and to promote alphabeta assembly (Lemas, M. V., Hamrick, M., Takeyasu, K., and Fambrough, D. M. (1994) J. Biol. Chem. 269, 8255-8259) When this 26-amino acid sequence of the rat Na,K-ATPase alpha3 subunit was replaced by the corresponding sequence of the rat gastric H,K-ATPase alpha subunit, the chimeric alpha subunit assembled preferentially with the rat gastric H,K-ATPase beta subunit (Wang, S.-G., Eakle, K. A., Levenson, R., and Farley, R. A. (1997) Am. J. Physiol. 272, C923-C930). In the present study, these 26 amino acids (Asn886-Ala911) of rat Na,K-ATPase alpha3 were replaced by the corresponding amino acids Asn908-Ala933 of rat distal colon H, K-ATPase. Site-directed mutagenesis of the chimeric alpha subunits and Na,K-ATPase alpha3 showed that Val904, Tyr898, and Cys908 in the Na,K-ATPase alpha3 subunit are key residues in alphabeta subunit interactions. The V904Q mutation in Na,K-ATPase alpha3 reduced the Bmax for ouabain binding and the ATPase activity of alpha3beta1 complexes by approximately 95%, and Y898R reduced the Bmax and ATPase activity by approximately 60%. The complementary mutations Q904V and R898Y increased the amount of ouabain bound by yeast membranes expressing the chimera with the colon H,K-ATPase sequence. The amount of ouabain bound by complexes assembled between Na, K-ATPase alpha3 containing the Y898R,C908G mutations and gastric H, K-ATPase beta was less than 10% of wild type Na,K-ATPase alpha3 expressed with the same beta subunit. The R898Y,G908C mutations in the chimeric alpha subunits also increased ouabain binding.
Wang
S G
SG
Department of Physiology and Biophysics, University of Southern California School of Medicine, Los Angeles, California 90033, USA.
Farley
R A
RA
D000818:Animals; D003545:Cysteine / Q000378:metabolism*; D004795:Enzyme Stability; D006358:Hot Temperature; D016297:Mutagenesis, Site-Directed; D010042:Ouabain / Q000378:metabolism; D011485:Protein Binding; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D000254:Sodium-Potassium-Exchanging ATPase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014443:Tyrosine / Q000378:metabolism*; D014633:Valine / Q000378:metabolism*
D011994:Recombinant Proteins; D014443:Tyrosine; D010042:Ouabain; D000254:Sodium-Potassium-Exchanging ATPase; D014633:Valine; D003545:Cysteine