Purification of Na+,K+-ATPase expressed in Pichia pastoris reveals an essential role of phospholipid-protein interactions

Abstract

Na+,K+-ATPase (porcine α/his 10-β) has been expressed in Pichia Pastoris, solubilized in n-dodecyl-β-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 °C. Both recombinant Na +,K+-ATPase and native pig kidney Na+,K +-ATPase, dissolved in n-dodecyl-β-maltoside, appear to be mainly stable monomers (α/β) as judged by size exclusion chromatography and sedimentation velocity. Na+,K+-ATPase activities at 37 °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 β subunit is expressed in Pichia Pastoris as two lightly glycosylated polypeptides and is quantitatively deglycosylated by endoglycosidase-H at 0 °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 β subunit in stabilizing conformations of Na+,K +-ATPase (or N+,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. © 2005 by The American Society for Biochemistry and Molecular Biology, Inc.