A Conserved Asparagine in a P-type Proton Pump Is Required for Efficient Gating of Protons

Kira Ekberg,Alex G Wielandt,Morten J Buch-Pedersen,Michael G Palmgren

doi:10.1074/jbc.m112.417345

Kira Ekberg, Alex G Wielandt + Show 2 more

Open Access

https://doi.org/10.1074/jbc.m112.417345

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Abstract

The minimal proton pumping machinery of the Arabidopsis thaliana P-type plasma membrane H(+)-ATPase isoform 2 (AHA2) consists of an aspartate residue serving as key proton donor/acceptor (Asp-684) and an arginine residue controlling the pKa of the aspartate. However, other important aspects of the proton transport mechanism such as gating, and the ability to occlude protons, are still unclear. An asparagine residue (Asn-106) in transmembrane segment 2 of AHA2 is conserved in all P-type plasma membrane H(+)-ATPases. In the crystal structure of the plant plasma membrane H(+)-ATPase, this residue is located in the putative ligand entrance pathway, in close proximity to the central proton donor/acceptor Asp-684. Substitution of Asn-106 resulted in mutant enzymes with significantly reduced ability to transport protons against a membrane potential. Sensitivity toward orthovanadate was increased when Asn-106 was substituted with an aspartate residue, but decreased in mutants with alanine, lysine, glutamine, or threonine replacement of Asn-106. The apparent proton affinity was decreased for all mutants, most likely due to a perturbation of the local environment of Asp-684. Altogether, our results demonstrate that Asn-106 is important for closure of the proton entrance pathway prior to proton translocation across the membrane.

Highlights

P-type proton pumps work against large electrochemical gradients, but how gating is controlled is poorly understood
Mutations at Asn-106 were introduced in a truncated version of AHA2 lacking 73 C-terminal residues comprising most of its regulatory domain and thereby rendered constitutively active [20, 29]
Mutations at Asn-106 Abolish the Ability of the Plant PM Hϩ-ATPase to Complement pma1—When situated in the plasma membrane of a living cell, the PM Hϩ-ATPase faces the challenge of pumping protons against a steep electrochemical gradient involving both a pH gradient and a membrane potential

Summary

Introduction

P-type proton pumps work against large electrochemical gradients, but how gating is controlled is poorly understood. Results: Substitution of Asn-106 in transmembrane segment M2 significantly reduces the ability of the pump to perform uphill proton transport. The minimal proton pumping machinery of the Arabidopsis thaliana P-type plasma membrane H؉-ATPase isoform 2 (AHA2) consists of an aspartate residue serving as key proton donor/acceptor (Asp-684) and an arginine residue controlling the pKa of the aspartate. Other important aspects of the proton transport mechanism such as gating, and the ability to occlude protons, are still unclear. An asparagine residue (Asn-106) in transmembrane segment 2 of AHA2 is conserved in all P-type plasma membrane H؉-ATPases. In the crystal structure of the plant plasma membrane H؉-ATPase, this residue is located in the putative ligand entrance pathway, in close proximity to the central proton donor/acceptor Asp-684. Substitution of Asn-106 resulted in mutant enzymes with significantly reduced ability to transport protons against a membrane potential. Our results demonstrate that Asn-106 is important for closure of the proton entrance pathway prior to proton translocation across the membrane

Methods

Results

Conclusion