Potassium as an Intrinsic Uncoupler of the Plasma Membrane H+-ATPase

Torben S Berner,Kees Venema,Elena L Rudashevskaya,Morten J Buch-Pedersen,Michael G Palmgren

doi:10.1074/jbc.m604781200

Abstract

The plant plasma membrane proton pump (H(+)-ATPase) is stimulated by potassium, but it has remained unclear whether potassium is actually transported by the pump or whether it serves other roles. We now show that K(+) is bound to the proton pump at a site involving Asp(617) in the cytoplasmic phosphorylation domain, from where it is unlikely to be transported. Binding of K(+) to this site can induce dephosphorylation of the phosphorylated E(1)P reaction cycle intermediate by a mechanism involving Glu(184) in the conserved TGES motif of the pump actuator domain. Our data identify K(+) as an intrinsic uncoupler of the proton pump and suggest a mechanism for control of the H(+)/ATP coupling ratio. K(+)-induced dephosphorylation of E(1)P may serve regulatory purposes and play a role in negative regulation of the transmembrane electrochemical gradient under cellular conditions where E(1)P is accumulating.

Highlights

During turnover, P-type ATPases alternate between different conformations that have very different structures
The D684N mutant of plasma membrane Hϩ-ATPase is Hϩ transport-incompetent but can hydrolyze ATP and, as it is defective in the E1P–E2P conformational step, E1P will start to accumulate [19, 20]
As no inhibitors of E1P–E2P partial reactions are known for plasma membrane Hϩ-ATPases, the D684N mutant serves as a unique tool to investigate partial reactions involving E1P

Summary

EXPERIMENTAL PROCEDURES

Site-directed Mutagenesis—The construction of the wildtype Hϩ-ATPase vector for heterologous expression in the yeast Saccharomyces cerevisiae, as well as the construction of the D684N mutant, has been described [19]. Affinity purification of heterologously expressed wild-type and D684N mutant Hϩ-ATPases was performed as described [20] except that purified proteins after purification were dialyzed against GMED20 (20% (w/v) glycerol, 100 mM MES2 (pH 6.5 with N-methyl-Dglucamine), 1 mM EDTA, and 1 mM dithiothreitol). Measurement of ATPase activity at 1 ␮M ATP was performed at 0 °C in a buffer (300 ␮l) containing 20 mM MOPS (pH adjusted to 6.5 with N-methyl-D-glucamine), 1 mM MgSO4, and purified protein in reactivation buffer and various cations as indicated. Reconstitution of Hϩ-ATPases into Artificial Liposomes—To determine the sidedness of the Kϩ effect, protein was reconstituted at a lipid to protein ratio of 200:1 as described [22] in 10 mM MES (adjusted to pH 6.5 with N-methyl-D-glucamine), 50 mM Na2SO4 or 50 mM K2SO4, and 20% (v/v) glycerol. Protein Determination—Protein concentrations were determined by the method of Bradford [25] employing bovine serum albumin as a standard

RESULTS

ATPase activitya

DISCUSSION