Abstract
The mechanism of proton pumping by P-type H(+)-ATPases is still unclear. In the plant P-type plasma membrane H(+)-ATPase AHA2, two charged residues, Arg(655) and Asp(684), are conserved in transmembrane segments M5 and M6, respectively, a region that has been shown be contribute to ion coordination in related P-type ATPases. Substitution of Arg(655) with either alanine or aspartate resulted in mutant enzymes exhibiting a significant shift in the P-type ATPase E(1)P-E(2)P conformational equilibrium. The mutant proteins accumulated in the E(1)P conformation, but were capable of conducting proton transport. This points to an important role of Arg(655) in the E(1)P-E(2)P conformational transition. The presence of a carboxylate moiety at position Asp(684) proved essential for coupling between initial proton binding and proton pumping. The finding that the carboxylate side chain of Asp(684) contributes to the proton-binding site and appears to function as an absolutely essential proton acceptor along the proton transport pathway is discussed in the context of a possible proton pumping mechanism of P-type H(+)-ATPases.
Highlights
An essential protein of the plant plasma membrane is the Hϩ-ATPase
To test whether any of the plant plasma membrane Hϩ-ATPase AHA2 substitutions were able to complement a pma1 mutation, each mutant was expressed in the yeast strain RS-72, in which the constitutive promoter of the PMA1 gene has been replaced by the galactose-dependent GAL1 promoter at the chromosomal level [23]
The goal of the present study was to determine the role in proton transport of conserved residues Arg655 and Asp684 situated in the middle of transmembrane segments M5 and M6, respectively, of the plant plasma membrane Hϩ-ATPase
Summary
An essential protein of the plant plasma membrane is the Hϩ-ATPase. It is a major protein at this membrane, where it controls cytoplasmic and apoplastic pH by generating the transmembrane electrochemical proton gradient that serves as a driving force for nutrient uptake [1, 2]. Protons in solution (as H3Oϩ) form solvation structures characteristic of cationic solvation This suggests that the plant Hϩ-ATPase possibly could mediate hydronium ion transport [17]. We investigated whether two charged residues, Arg655AHA2 and Asp684AHA2, situated in transmembrane segments M5 and M6, respectively, might play a role in the proton pumping mechanism of plasma membrane Hϩ-ATPase. During proteolysis of the related Naϩ/Kϩ- and Hϩ/Kϩ-ATPases, inclusion of potassium ions stabilizes the M5M6 hairpin loop, and it has been suggested that this moiety moves in and out of the membrane during catalysis [18, 19] It is evident from the crystal structures of SERCA1 Ca2ϩ-ATPase in two conformations [7, 9] that the M5M6 segment is a mobile structure, somehow coupling.
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