Abstract
The plasma membrane H(+)-ATPase AHA2 of Arabidopsis thaliana, which belongs to the P-type ATPase superfamily of cation-transporting ATPases, pumps protons out of the cell. To investigate the mechanism of ion transport by P-type ATPases we have mutagenized Asp(684), a residue in transmembrane segment M6 of AHA2 that is conserved in Ca(2+)-, Na(+)/K(+)-, H(+)/K(+)-, and H(+)-ATPases and which coordinates Ca(2+) ions in the SERCA1 Ca(2+)-ATPase. We describe the expression, purification, and biochemical analysis of the Asp(684) --> Asn mutant, and provide evidence that Asp(684) in the plasma membrane H(+)-ATPase is required for any coupling between ATP hydrolysis, enzyme conformational changes, and H(+)-transport. Proton pumping by the reconstituted mutant enzyme was completely abolished, whereas ATP was still hydrolyzed. The mutant was insensitive to the inhibitor vanadate, which preferentially binds to P-type ATPases in the E(2) conformation. During catalysis the Asp(684) --> Asn enzyme accumulated a phosphorylated intermediate whose stability was sensitive to addition of ADP. We conclude that the mutant enzyme is locked in the E(1) conformation and is unable to proceed through the E(1)P-E(2)P transition.
Highlights
P-type ATPases are relatively small ion pumps with a single catalytic subunit, and are characterized by forming a phosphorylated ( P-type) intermediate during the reaction cycle [1]
We suggest a fundamental role for Asp684 in coordinating coupling of ATP hydrolysis to Hϩ-transport in the plant plasma membrane Hϩ-ATPase
The COOH terminus of the plant plasma membrane Hϩ-ATPase AHA2 is a regulatory domain containing autoinhibitory residues [26], and removal of this domain, which was done at the gene level, renders the enzyme constitutively active [21]
Summary
P-type ATPases are relatively small ion pumps with a single catalytic subunit, and are characterized by forming a phosphorylated ( P-type) intermediate during the reaction cycle [1]. Cryoelectron microscopic images of the Hϩ-ATPase of Neurospora crassa [4] have been obtained at 8-Å resolution, and the crystal structure of the sarcoplasmic reticulum Ca2ϩ-ATPase [5] has recently been solved at 2.6-Å resolution [6] These structures reveal the presence of 10 transmembrane ␣-helices connected to a large cytoplasmic region of the enzyme. A large amount of biochemical data support this model [8, 9], and it is likely that one or more transmembrane helices in concert form the ion-binding site(s) in other related P-type ATPases. Mutagenesis studies have identified a number of amino acid residues within M4, M5, and M6 of the Naϩ/Kϩ-ATPase that appear to be critical for cation binding [11,12,13,14].
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