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

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

doi:10.1074/jbc.m414290200

Abstract

Na+,K+-ATPase (porcine alpha/his10-beta) has been expressed in Pichia Pastoris, solubilized in n-dodecyl-beta-maltoside and purified to 70-80% purity by nickel-nitrilotriacetic acid chromatography combined with size exclusion chromatography. The recombinant protein is inactive if the purification is done without added phospholipids. The neutral phospholipid, dioleoylphosphatidylcholine, preserves Na+,K+-ATPase activity of protein prepared in a Na+-containing medium, but activity is lost in a K+-containing medium. By contrast, the acid phospholipid, dioleoylphosphatidylserine, preserves activity in either Na+- or K+-containing media. In optimal conditions activity is preserved for about 2 weeks at 0 degrees C. Both recombinant Na+,K+-ATPase and native pig kidney Na+,K+-ATPase, dissolved in n-dodecyl-beta-maltoside, appear to be mainly stable monomers (alpha/beta) as judged by size exclusion chromatography and sedimentation velocity. Na+,K+-ATPase activities at 37 degrees C of the size exclusion chromatography-purified recombinant and renal Na+,K+-ATPase are comparable but are lower than that of membrane-bound renal Na+,K+-ATPase. The beta subunit is expressed in Pichia Pastoris as two lightly glycosylated polypeptides and is quantitatively deglycosylated by endoglycosidase-H at 0 degrees C, to a single polypeptide. Deglycosylation inactivates Na+,K+-ATPase prepared with dioleoylphosphatidylcholine, whereas dioleoylphosphatidylserine protects after deglycosylation, and Na+,K+-ATPase activity is preserved. This work demonstrates an essential role of phospholipid interactions with Na+,K+-ATPase, including a direct interaction of dioleoylphosphatidylserine, and possibly another interaction of either the neutral or acid phospholipid. Additional lipid effects are likely. A role for the beta subunit in stabilizing conformations of Na+,K+-ATPase (or H+,K+-ATPase) with occluded K+ ions can also be inferred. Purified recombinant Na+,K+-ATPase could become an important experimental tool for various purposes, including, hopefully, structural work.

Highlights

The Naϩ,Kϩ-ATPase is a member of the P-type ATPase family of active cation pumps
Na؉,K؉-ATPase has been expressed in Pichia Pastoris, solubilized in n-dodecyl-␤-maltoside and purified to 70 – 80% purity by nickel-nitrilotriacetic acid chromatography combined with size exclusion chromatography
The neutral phospholipid, dioleoylphosphatidylcholine, preserves Na؉,K؉-ATPase activity of protein prepared in a Na؉-containing medium, but activity is lost in a K؉-containing medium

Summary

EXPERIMENTAL PROCEDURES

Materials—P. pastoris protease-deficient strain SMD1165 (his, prb1) was used for transformation. 1 ml of beads was washed with 10 ml of buffers containing NaCl, 250 mM; Tris1⁄7HCl, 20 mM, pH 7.4; DDM, 0.2 mg/ml; DOPS (or other combinations of lipid), 0.05 mg/ml; glycerol, 10%; and imidazole, 30 mM (first wash) or imidazole, 60 mM (second wash). Apparent Mr values of the eluted Naϩ,Kϩ-ATPase were calculated from the equation: Elution volume ϭ A*log Mr ϩ B, where A ϭ Ϫ4.681 Ϯ 0.315 and B ϭ 22.77 Ϯ 0.723, obtained by linear regression of data on elution of standard soluble proteins. Binding of [14C]DDM—[14C]DDM was added to about 100 ␮g of the HPLC peak 2 of the pig kidney Naϩ,Kϩ-ATPase, or to elution buffer without protein, and concentrated by centrifugation on a Centricon 100 ultrafiltration filter. Naϩ,Kϩ-ATPase activities were assayed at 0 °C using [␥-32P]ATP, routinely at 2 ␮M final

Turnover number

RESULTS

Mb nm

DISCUSSION