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

Abstract

؉ -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 3 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. P-type ATPases are relatively small ion pumps with a single catalytic subunit, and are characterized by forming a phosphorylated (hence P-type) intermediate during the reaction cycle (1). Plant plasma membrane H ϩ -ATPases belong to the subgroup of P 3 -type ATPases, which comprises proton pumps and is closely related to the subgroup of P 2 -type ATPases, which includes cation pumps such as the mammalian Na ϩ /K ϩ -, Ca 2ϩ -, and H ϩ

The Arabidopsis Na+/H+Exchanger AtNHX1 Catalyzes Low Affinity Na+ and K+ Transport in Reconstituted Liposomes

“…Reconstitution of AtNHX1:RGSH 6 into Vesicles-For reconstitution of purified AtNHX1:RGSH 6 into artificial membranes, essentially the same protocol was used as previously developed for reconstitution of the purified His-tagged plasma membrane proton ATPase AHA2 (23). Protein (4 g) was mixed with soybean phospholipids type II-S (Sigma) at a lipid to protein ratio of 585 in a total volume of 208 l of reconstitution buffer containing 20 mM BTP-MES, pH 7.5, 10% glycerol, 25 mM (NH 4 ) 2 SO 4 , and 2.5 mM pyranine (converted to BTP salt using Dowex 50WX8 ion exchange resin).…”

“…500 l of this preculture was transferred to 600 ml of SDA-galactose and grown at 30°C to saturation (A 660 nm ϭ 5.0). This culture was then inoculated into 6 liters of YPD medium and grown for 20 h. The cells were harvested, and the microsomes were isolated as described (23). The microsomal membrane fraction (4 ml, 5 mg protein/ml) was mixed with 20 ml of solubilization buffer (50 mM KH 2 PO 4 , pH 7.4, 500 mM NaCl, 10 mM imidazole, 20% glycerol, 0.5% n-dodecyl-␤-D-maltoside, 0.2 mM phenylmethylsulfonyl fluoride, 10 g/ml chymostatin, 2 g/ml pepstatin) and incubated for 30 min at 4°C under gentle shaking.…”

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

“…The modified cDNA encodes the AHA2 PM H ϩ -ATPase with a C-terminal deletion of 73 amino acid residues and the insertion of a C-terminal Met-Arg-Gly-Ser-His 6 (MRGSH 6 ) tag (20). The C-terminal deletion renders the PM H ϩ -ATPase constitutively active, whereas the addition of the His 6 tag allows for affinity purification of the constructed mutants.…”

“…Purification and Reconstitution of ATPase-Growth of yeast for protein purification was the same as described (20). The transformed yeast cells were harvested, and the PM H ϩ -ATPases were purified by membrane solubilization using n-dodecyl-␤-D-maltoside and Ni 2ϩ -nitrilotriacetic acid affinity chromatography according to established procedures (12).…”

“…Diverse proton transporters such as bacteriorhodopsin, the F O F 1 ATP synthase, and the PM H ϩ -ATPase appear to employ a common proton transport mechanism that involves a central aspartate residue serving as key proton donor/acceptor and an arginine residue controlling the pK a of that aspartate residue as the minimal proton pumping apparatus (19). For the plant PM H ϩ -ATPase, the central proton donor/acceptor has been shown to be the essential residue Asp-684, and, in addition, the nearby Arg-655 has been shown to be important, although not essential, for proton transport in this protein (20,21). In addition to the involvement of these two residues and the recent identification of a proton release group (22), not much is known about how proton transport is achieved by PM H ϩ -ATPases.…”