Abolishment of proton pumping and accumulation in the E1P conformational state of a plant plasma membrane H+-ATPase by substitution of a conserved aspartyl residue in transmembrane segment 6.
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.
Buch-Pedersen
M J
MJ
Department of Plant Biology, The Royal Danish Veterinary and Agricultural University, Thorvaldsensvej 40, DK-1871 Frederiksberg C, Denmark.
Venema
K
K
Serrano
R
R
Palmgren
M G
MG
D000251:Adenosine Triphosphatases / Q000378:metabolism; D017360:Arabidopsis / Q000201:enzymology; D001216:Asparagine / Q000737:chemistry; D001224:Aspartic Acid / Q000737:chemistry; D015153:Blotting, Western; D002384:Catalysis; D002462:Cell Membrane / Q000201:enzymology*; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007700:Kinetics; D008024:Ligands; D016297:Mutagenesis, Site-Directed; D010766:Phosphorylation; D010940:Plant Proteins / Q000737:chemistry*; D011487:Protein Conformation; D006180:Proton-Translocating ATPases / Q000378:metabolism; D011522:Protons; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D013997:Time Factors; D014357:Trypsin / Q000494:pharmacology; D014638:Vanadates / Q000494:pharmacology
D008024:Ligands; D010940:Plant Proteins; D011522:Protons; D001224:Aspartic Acid; D014638:Vanadates; D001216:Asparagine; D014357:Trypsin; D000251:Adenosine Triphosphatases; D006180:Proton-Translocating ATPases