title: A conserved asparagine in a P-type proton pump is required for efficient gating of protons.
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.
authors: Department of Plant and Environmental Sciences, Centre for Membrane Pumps in Cells and Disease-PUMPKIN, Danish National Research Foundation, University of Copenhagen, Thorvaldsensvej 40, DK-1871 Frederiksberg, Denmark.
authors: Kira
authors: K
authors: Ekberg
authors: Department of Plant and Environmental Sciences, Centre for Membrane Pumps in Cells and Disease-PUMPKIN, Danish National Research Foundation, University of Copenhagen, Thorvaldsensvej 40, DK-1871 Frederiksberg, Denmark.
authors: Alex G
authors: AG
authors: Wielandt
authors: Department of Plant and Environmental Sciences, Centre for Membrane Pumps in Cells and Disease-PUMPKIN, Danish National Research Foundation, University of Copenhagen, Thorvaldsensvej 40, DK-1871 Frederiksberg, Denmark.
authors: Morten J
authors: MJ
authors: Buch-Pedersen
authors: Department of Plant and Environmental Sciences, Centre for Membrane Pumps in Cells and Disease-PUMPKIN, Danish National Research Foundation, University of Copenhagen, Thorvaldsensvej 40, DK-1871 Frederiksberg, Denmark. Electronic address: palmgren@life.ku.dk.
authors: Michael G
authors: MG
authors: Palmgren
mesh_terms: D000251:Adenosine Triphosphatases / Q000737:chemistry; D017360:Arabidopsis / Q000378:metabolism*; D029681:Arabidopsis Proteins / Q000737:chemistry* / Q000502:physiology*; D001120:Arginine / Q000737:chemistry; D001216:Asparagine / Q000737:chemistry* / Q000235:genetics; D001692:Biological Transport; D002462:Cell Membrane / Q000201:enzymology; D018360:Crystallography, X-Ray / Q000379:methods; D003600:Cytosol / Q000378:metabolism; D004247:DNA / Q000235:genetics; D004563:Electrochemistry / Q000379:methods; D018506:Gene Expression Regulation, Plant; D005816:Genetic Complementation Test; D006863:Hydrogen-Ion Concentration; D008564:Membrane Potentials; D008958:Models, Molecular; D009154:Mutation; D011401:Promoter Regions, Genetic; D011487:Protein Conformation; D017494:Proton Pumps / Q000502:physiology*; D006180:Proton-Translocating ATPases / Q000737:chemistry* / Q000502:physiology*; D011522:Protons; D012441:Saccharomyces cerevisiae / Q000235:genetics
chemical_list: D029681:Arabidopsis Proteins; D017494:Proton Pumps; D011522:Protons; D001216:Asparagine; D004247:DNA; D001120:Arginine; D000251:Adenosine Triphosphatases; D006180:Proton-Translocating ATPases; C479903:AHA2 protein, Arabidopsis