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Mutation of a conserved proline residue in the beta-subunit ectodomain prevents Na(+)-K(+)-ATPase oligomerization
K Geering1, P Jaunin, F Jaisser
1Institut de Pharmacologie et de Toxicologie de l'Université, Lausanne, Switzerland.
Abstract:
A highly conserved sequence motif (4 tyrosines and 1 proline: YYPYY) of the Na(+)-K(+)-adenosinetriphosphatase (ATPase) beta 1-subunit ectodomain has been mutagenized to study its possible role in alpha/beta-assembly and sodium pump function. Single as well as double tyrosine mutants (tyrosine to phenylalanine: Y to F) of Xenopus laevis beta 1-subunits are able to associate with alpha 1-subunits and form functional Na-K pumps at the plasma membrane that are indistinguishable from wild-type alpha 1, beta 1-Na-K pumps (as assessed by measurements of ouabain binding, 86Rb flux, Na-K pump current, and activation by external potassium). In contrast, a single proline mutation (proline to glycine: P244G) reduced by > 90% the proper assembly and function of Na(+)-K(+)-ATPase, despite a normal rate of synthesis and core glycosylation. Our data indicate that proline-244 plays a critical role in the proper folding of the beta-subunit and its ability to associate efficiently with the alpha 1-subunit in the endoplasmic reticulum.
Insights
The proline at position 244 in the Na(+)-K(+)-ATPase beta 1-subunit is crucial for proper folding and assembly with the alpha 1-subunit. Tyrosine mutations do not affect sodium pump function or assembly.
Area of Science:
- Biochemistry
- Molecular Biology
- Cell Physiology
Background:
- The Na(+)-K(+)-adenosinetriphosphatase (ATPase) is a vital ion pump essential for maintaining cellular homeostasis.
- The beta 1-subunit of Na(+)-K(+)-ATPase contains a conserved YYPYY motif in its ectodomain, implicated in subunit assembly and pump function.
Purpose of the Study:
- To investigate the role of the conserved YYPYY motif in the Na(+)-K(+)-ATPase beta 1-subunit.
- To determine the specific contributions of tyrosine and proline residues within this motif to alpha/beta-subunit assembly and sodium pump activity.
Main Methods:
- Site-directed mutagenesis was employed to create tyrosine (to phenylalanine) and proline (to glycine) mutants of the Xenopus laevis beta 1-subunit.
- Functional assessment of Na(+)-K(+)-ATPase activity was performed using ouabain binding, 86Rb flux measurements, and Na-K pump current analysis.
- Subunit assembly was evaluated by assessing the association of alpha and beta subunits.
Main Results:
- Single and double tyrosine-to-phenylalanine mutations in the beta 1-subunit did not impair alpha/beta-subunit association or the formation of functional Na-K pumps.
- The proline-to-glycine mutation at position 244 (P244G) drastically reduced proper Na(+)-K(+)-ATPase assembly and function by over 90%.
- The P244G mutation did not affect the synthesis rate or core glycosylation of the beta 1-subunit.
Conclusions:
- Proline-244 is critical for the correct folding of the Na(+)-K(+)-ATPase beta 1-subunit.
- Efficient association of the beta 1-subunit with the alpha 1-subunit in the endoplasmic reticulum is dependent on proline-244.
- The tyrosine residues in the YYPYY motif are not essential for Na(+)-K(+)-ATPase assembly or function, suggesting a specialized role for proline-244.