Purification of Na+,K+-ATPase Expressed in Pichia pastoris Reveals an Essential Role of Phospholipid-Protein Interactions

Cohen, Eytan; Goldshleger, Rivka; Shainskaya, Alla; Tal, Daniel M.; Ebel, Christine; Maire, Marc le; Karlish, Steven J. D.

doi:10.1074/jbc.m414290200

Cited by 70 publications

(120 citation statements)

References 58 publications

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“…In relation to the lipid content of the preparations, preliminary shotgun mass spectrometry analyses of the lipids in the preparations did not reveal species other than those added to the preparations. 5 Lipid Specificity of Stimulation of NaK-ATPase-As shown recently, unsaturated neutral phospholipids (PC or PE, especially 18:2/18:2 PC and 18:2/18:2 PE) stimulate the specific Na,K-ATPase activity of human ␣1␤1 FXYD1 complexes by increasing the molar turnover rate (27,37). We have now investigated the selectivity of this effect more systematically using a selection of synthetic mono-, di-, and polyunsaturated PCs and PEs (Fig.…”

Section: Resultsmentioning

confidence: 99%

Stimulation, Inhibition, or Stabilization of Na,K-ATPase Caused by Specific Lipid Interactions at Distinct Sites

Habeck¹,

Haviv²,

Katz³

et al. 2015

Journal of Biological Chemistry

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Background: Na,K-ATPase is stabilized by phosphatidylserine/cholesterol and is stimulated by neutral phospholipids. Results: Three specific lipid-Na,K-ATPase interactions are detectable that either (a) stabilize the protein or (b) stimulate or (c) inhibit Na,K-ATPase activity, with distinct kinetic mechanisms. Conclusion: There are separate binding sites for phosphatidylserine/cholesterol (stabilizing), polyunsaturated phosphatidylethanolamine (stimulatory), and sphingomyelin/cholesterol (inhibitory). Significance: In physiological conditions, specifically bound lipids may regulate Na,K-ATPase activity.

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Section: Resultsmentioning

confidence: 99%

Stimulation, Inhibition, or Stabilization of Na,K-ATPase Caused by Specific Lipid Interactions at Distinct Sites

Habeck¹,

Haviv²,

Katz³

et al. 2015

Journal of Biological Chemistry

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“…37. D369N/D369A/D369E, T212A, and E214A/E214Q mutants were prepared by the methods described previously (39 clones was determined by Western blots, using the antibody anti-KETYY antibody (at a dilution of 1:3000), which recognizes the C terminus of the ␣ subunit.…”

Section: Methodsmentioning

confidence: 99%

“…Ouabain binding was also carried out to determine the highest expressing clones that were subsequently used routinely. For large scale growth and expression of the recombinant Na (20,37), single colonies propagated in YPD medium were inoculated into 5 ml of BMG liquid medium, which was incubated for 48 h with vigorous shaking at 30°C. The culture was further diluted 100-fold to a final volume of 300 ml and grown in the BMG medium (glycerol 1%) for a further 24 h to an A 600 of 2-6.…”

Section: Methodsmentioning

confidence: 99%

“…ϩ -ATPase-The methods have been described in detail (37,38,40). Briefly, unlabeled or FITC-labeled membranes (1-2 mg/ml) were homogenized well for 15 min on ice with DDM (DDM/protein, 2:1 w/w) in a medium containing 250 mM NaCl, 20 mM Tris-HCl, pH 7.4, 5 mM imidazole, 0.5 mM phenylmethylsulfonyl fluoride, and 10% glycerol.…”

Section: Solubilization and Purification Of Fitc-labeled Recombinant Namentioning

confidence: 99%

“…Recent publications from this laboratory describe expression of Na ϩ ,K ϩ -ATPase in P. pastoris and purification of the recombinant protein in a detergent-soluble functional state in quantities up to 1 mg (20,37,38). The high yield of purified protein now makes it possible to carry out detailed biochemical and biophysical work.…”

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Neutralization of the Charge on Asp369 of Na+,K+-ATPase Triggers E1 ↔ E2 Conformational Changes

Belogus

Haviv

Karlish

2009

Journal of Biological Chemistry

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favors disengagement of the A domain from N and P domains (E 1 ), whereas the neutral D369N/D369A mutants favor association of the A domain (TGES sequence) with P and N domains (E 2 ). Changes in charge interactions of Asp 369 may play an important role in triggering E 1 P(3Na) 7 E 2 P and E 2 (2K) 3 E 1 Na transitions in native Na ؉ ,K ؉ -ATPase.The kinetic mechanism of P-type cation pumps is now well established. Active cation transport involves covalent phosphorylation by ATP and dephosphorylation of an aspartate residue coupled to cation movements mediated by E 1 7 E 2 2 conformational changes. The molecular mechanism is, of course, the central question of energy transduction. Molecular structures of the sarcoplasmic reticulum Ca 2ϩ -ATPase (SERCA1) in several conformations are available (1-3). Two crystal structures of the Na ؉ ,K ؉ -ATPase, consisting of ␣, ␤, and FYXD subunits (4), in an E 2 (2Rb)-MgF 4 2Ϫ conformation (equivalent to E 2 (2Rb)⅐P) have been published recently (5, 6). The architecture of the ␣ subunit is very similar to the Ca 2ϩ -ATPase, consisting of head, stalk, and membrane sectors, with 10 trans-membrane segments and a cytoplasmic sector consisting of N (nucleotide binding), P (phosphorylating), and A (anchor or actuator) domains. Compared with the first published structure of the Na ϩ ,K ϩ -ATPase at 3.5 Å (5), the more recent structure at 2.4 Å (6) reveals greater detail of the cation binding domain, resolution of the ␤ subunit ectodomain, and an FXYD protein ectodomain.In the case of Na ϩ ,K ϩ -ATPase, three Na ؉ ions and two K ؉ ions are transported in the E 1 ATP 3 E 1 P(3Na) 3E 2 P and the E 2 P 3 E 2 (2K) 3 E 1 ATP halves of the cycle, respectively, at the expense of one molecule of ATP. Binding of ATP with low affinity to E 2 (2K) is the first step in the catalytic cycle and is followed by accelerated conversion of E 2 (2K)ATP to E 1 ⅐ATP in which ATP is bound with high affinity (7-9). In the case of Ca 2ϩ -ATPase, two Ca 2ϩ ions and two protons are transported in the E 1 ATP 3 E 1 P 3 E 2 P E 2 P 3 E 2 3 E 1 ATP halves of the cycle, respectively.The mechanism of energy transduction by P-type cation pumps is best addressed by reference to the Ca 2ϩ -ATPase, which has been crystallized in most of the relevant conformations, reviewed recently in detail (1-3). Crystal structures of Ca 2ϩ -ATPase show mainly rigid body movements of the N, P, and A domains, mediated by the flexible linkers between the N and P and between the A domain and trans-membrane segment (M1-M3), coupled mechanically to movements of M1-M6 trans-membrane segments that allow alternate access of two Ca 2ϩ ions and two protons to the occlusion sites within M4, M5, M6, and M8. M7-M10 segments appear to act as immovable anchors. In the E 1 ATP state, N and P domains are in close proximity cross-linked by the bound ATP, whereas the A domain is displaced to one side. Following phosphorylation to E 1 P, the conformational change to E 2 P involves a large rotation of the A domain bringing it into close proximity with th...

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The Sodium PotassiumATPase

Nyblom¹,

Morth²,

Nissen³

2004

Handbook of Metalloproteins

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At the expense of adenosine triphosphate (ATP), the P‐type ATPase family of membrane proteins actively transports different cations or lipids, in a reaction cycle that includes the phosphorylation/dephosphorylation of a conserved aspartate residue. These proteins share a common structural and mechanistic core, but Na + , K + ‐ATPase is the first multisubunit P‐type ATPase to be structurally determined. The Na + , K + ‐ATPase exports three Na + ‐ ions per ATP molecule from the cytoplasm, while at the same time transporting two K + ‐ ions in the opposite direction. This electrogenic pump is critical for human and animal cells where the gradients of these ions are important for maintaining the resting potential and for electrical excitability of e.g. muscle and nerve cells, and for energization of secondary transporters and osmotic control. This article reviews the currently available structural knowledge of the Na + , K + ‐ATPase. Hitherto, there are only structures of one of the catalytic forms reported representing the potassium‐occluded state ([K 2 ] E 2·P i ). The overall architecture of the αβγ‐complex is presented and the differences compared to the previously characterized structures of the sarco(endo)plasmic reticulum Ca 2+ ‐ATPase (SERCA) are pinpointed. Furthermore, the properties of the K + ‐ion binding sites are described and the functional relevance of the C‐terminus is addressed.

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Purification of Na+,K+-ATPase Expressed in Pichia pastoris Reveals an Essential Role of Phospholipid-Protein Interactions

Cited by 70 publications

References 58 publications

Stimulation, Inhibition, or Stabilization of Na,K-ATPase Caused by Specific Lipid Interactions at Distinct Sites

Stimulation, Inhibition, or Stabilization of Na,K-ATPase Caused by Specific Lipid Interactions at Distinct Sites

Neutralization of the Charge on Asp369 of Na+,K+-ATPase Triggers E1 ↔ E2 Conformational Changes

The Sodium PotassiumATPase

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