Hydrophobic C-terminal amino acids in the beta-subunit are involved in assembly with the alpha-subunit of Na,K-ATPase.
To define the structural basis of oligomerization for the alpha- and beta-subunits of Na,K-ATPase, we have attempted to identify the amino acids in the C-terminus of the beta-subunit that are involved in subunit assembly. We predicted that the last 10 amino acids form a beta-strand-like structure exposing on one side a hydrophilic and on the other side a continuous hydrophobic domain. The relative importance of the two domains in assembly was probed by introducing point mutations in either domain of Xenopus beta 3-subunits and by testing the ability of these mutants to stabilize newly synthesized alpha-subunits expressed in Xenopus oocytes and to form functional alpha-beta complexes at the plasma membrane. All single and double mutants with changes at R268 and/or K272 to either uncharged or negatively charged amino acids associated with coexpressed alpha-subunits and increased the number of ouabain binding sites and Rb uptake into oocytes. On the other hand, mutations affecting the hydrophobic amino acids influenced the assembly efficiency with alpha-subunits to a variable extent. The single mutants V269N and I275N did not influence and the mutant V273N slightly affected the assembly process. On the other hand, the cellular accumulation of alpha-subunits and the expression of functional Na,K pumps was considerably reduced with the mutant F271N and totally abolished with the double mutant V269N/F271N. Finally, replacement of V269 and F271 or V273 and I275 with the less hydrophobic alanine also significantly decreased subunit assembly, which was no longer detectable after replacement of all four amino acids.(ABSTRACT TRUNCATED AT 250 WORDS)
Beggah
A T
AT
Institute of Pharmacology and Toxicology, University of Lausanne, Switzerland.
Beguin
P
P
Jaunin
P
P
Peitsch
M C
MC
Geering
K
K
D000595:Amino Acid Sequence; D000818:Animals; D006801:Humans; D046911:Macromolecular Substances; D008845:Microinjections; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009865:Oocytes / Q000201:enzymology; D010042:Ouabain / Q000378:metabolism; D017433:Protein Structure, Secondary; D011817:Rabbits; D051381:Rats; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D000254:Sodium-Potassium-Exchanging ATPase / Q000737:chemistry*; D013329:Structure-Activity Relationship; D014982:Xenopus laevis
D046911:Macromolecular Substances; D010042:Ouabain; D000254:Sodium-Potassium-Exchanging ATPase