Abstract
FXYD proteins are a family of seven small regulatory proteins, expressed in a tissue-specific manner, that associate with Na,K-ATPase as subsidiary subunits and modulate kinetic properties. This study describes an additional property of FXYD proteins as stabilizers of Na,K-ATPase. FXYD1 (phospholemman), FXYD2 (γ subunit), and FXYD4 (CHIF) have been expressed in Escherichia coli and purified. These FXYD proteins associate spontaneously in vitro with detergent-soluble purified recombinant human Na,K-ATPase (α1β1) to form α1β1FXYD complexes. Compared with the control (α1β1), all three FXYD proteins strongly protect Na,K-ATPase activity against inactivation by heating or excess detergent (C(12)E(8)), with effectiveness FXYD1 > FXYD2 ≥ FXYD4. Heating also inactivates E(1) ↔ E(2) conformational changes and cation occlusion, and FXYD1 protects strongly. Incubation of α1β1 or α1β1FXYD complexes with guanidinium chloride (up to 6 m) causes protein unfolding, detected by changes in protein fluorescence, but FXYD proteins do not protect. Thus, general protein denaturation is not the cause of thermally mediated or detergent-mediated inactivation. By contrast, the experiments show that displacement of specifically bound phosphatidylserine is the primary cause of thermally mediated or detergent-mediated inactivation, and FXYD proteins stabilize phosphatidylserine-Na,K-ATPase interactions. Phosphatidylserine probably binds near trans-membrane segments M9 of the α subunit and the FXYD protein, which are in proximity. FXYD1, FXYD2, and FXYD4 co-expressed in HeLa cells with rat α1 protect strongly against thermal inactivation. Stabilization of Na,K-ATPase by three FXYD proteins in a mammalian cell membrane, as well the purified recombinant Na,K-ATPase, suggests that stabilization is a general property of FXYD proteins, consistent with a significant biological function.
Highlights
The Na,K-pump or Na,K-ATPase consists of a catalytic ␣ subunit, with 10 trans-membrane segments, and a glycosylated
FXYD1 expressed in P. pastoris associates spontaneously with either the human ␣1-porcine 1 or human ␣2-porcine 1 complexes of Na,K-ATPase bound to BD Talon beads to form the ␣FXYD1 complex, which is strongly protected against thermal inactivation [27, 28]
In the NaCl-containing medium (Fig. 2A), the purified human FXYD1 protected human ␣11 strongly against thermal inactivation at 45 °C, similar to the effect of unpurified FXYD1 expressed in P. pastoris
Summary
Purification of Recombinant FXYD Proteins and TEV Protease Treatment—Bacterial membranes suspended to a final concentration of 4 mg/ml in the medium containing 200 mM NaCl, 50 mM Tricine/Tris, pH 7.4, 10 mM imidazole, 0.5 mM phenylmethylsulfonyl fluoride (PMSF), 20% (v/v) glycerol were homogenized (glass-Teflon) with a solution containing 8 mg/ml n-dodecyl -maltoside (i.e. final DDM, 2:1 mg/mg protein). The FXYD proteins were eluted by mixing the beads at 4 °C with 1 bead volume of a solution containing 250 mM imidazole, 100 mM NaCl or KCl, or no salt, 50 mM Tricine/Tris, pH 7.4, 0.1 mg/ml C12E8, 0.1 mg/ml SOPS (or the indicated concentration), 0.01 mg/ml cholesterol, and 20% glycerol (“elution buffer”), with rotation for 1 h. The membranes were heated at 45 °C or not heated, and Naϩ,Kϩ-ATPase activity was measured with and without 10 mM ouabain in the reaction mixture
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