Kees Venema

Plant NHX cation/proton antiporters

Although physiological and biochemical data since long suggested that Na+/H+ and K+/H+ antiporters are involved in intracellular ion and pH regulation in plants, it has taken a long time to identify genes encoding antiporters that could fulfil these roles. Genome sequencing projects have now shown that plants contain a very large number of putative Cation/Proton antiporters, the function of which is only beginning to be studied. The intracellular NHX transporters constitute the first Cation/Proton exchanger family studied in plants. The founding member, AtNHX1, was identified as an important salt tolerance determinant and suggested to catalyze Na+ accumulation in vacuoles. It is, however, becoming increasingly clear, that this gene and other members of the family also play crucial roles in pH regulation and K+ homeostasis, regulating processes from vesicle trafficking and cell expansion to plant development.

Potassium as an intrinsic uncoupler of the plasma membrane H+-ATPase

The plant plasma membrane proton pump (H+-ATPase) is stimulated by potassium, but it has remained unclear whether potassium is actually transported by the pump or whether it serves other roles. We now show that K+ is bound to the proton pump at a site involving Asp617 in the cytoplasmic phosphorylation domain, from where it is unlikely to be transported. Binding of K+ to this site can induce dephosphorylation of the phosphorylated E1P reaction cycle intermediate by a mechanism involving Glu184 in the conserved TGES motif of the pump actuator domain. Our data identify K+ as an intrinsic uncoupler of the proton pump and suggest a mechanism for control of the H+/ATP coupling ratio. K+-induced dephosphorylation of E1P may serve regulatory purposes and play a role in negative regulation of the transmembrane electrochemical gradient under cellular conditions where E1P is accumulating.

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 Asp684, a residue in transmembrane segment M6 of AHA2 that is conserved in Ca2+-, Na+/K+-, H+/K+-, and H+-ATPases and which coordinates Ca2+ ions in the SERCA1 Ca2+-ATPase. We describe the expression, purification, and biochemical analysis of the Asp684 → Asn mutant, and provide evidence that Asp684 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 Asp684 → Asn enzyme accumulated a phosphorylated intermediate whose stability was sensitive to addition of ADP. We conclude that the mutant enzyme is locked in theE 1 conformation and is unable to proceed through the E 1P-E 2P transition.

Vacuolar cation/H+ antiporters of Saccharomyces cerevisiae

We previously demonstrated that Saccharomyces cerevisiae vnx1Δ mutant strains displayed an almost total loss of Na+ and K+/H+ antiporter activity in a vacuole-enriched fraction. However, using different in vitro transport conditions, we were able to reveal additional K+/H+ antiporter activity. By disrupting genes encoding transporters potentially involved in the vnx1 mutant strain, we determined that Vcx1p is responsible for this activity. This result was further confirmed by complementation of the vnx1Δvcx1Δ nhx1Δ triple mutant with Vcx1p and its inactivated mutant Vcx1p-H303A. Like the Ca2+/H+ antiporter activity catalyzed by Vcx1p, the K+/H+ antiporter activity was strongly inhibited by Cd2+ and to a lesser extend by Zn2+. Unlike as previously observed for NHX1 or VNX1, VCX1 overexpression only marginally improved the growth of yeast strain AXT3 in the presence of high concentrations of K+ and had no effect on hygromycin sensitivity. Subcellular localization showed that Vcx1p and Vnx1p are targeted to the vacuolar membrane, whereas Nhx1p is targeted to prevacuoles. The relative importance of Nhx1p, Vnx1p, and Vcx1p in the vacuolar accumulation of monovalent cations will be discussed.

P-type H(+)- and Ca(2+)-ATPases in plant cells

Plasma membrane H+-ATPase and Na+/K+-ATPase belong to the same subfamily of ion pumps that has been termed Pz-ATPases. I A phylogenetic tree showing the evolutionary relation between members of the P-type ATPase superfamily is presented in FIGURE 1. Plasma membrane proton pumps appear to constitute a monophyletic group of P-type ATPases and are present in plants, fungi, algae, protozoa, and archaea. This wide distribution among species suggests that P-type H+-ATPases appeared very early in evolution. On the contrary, Na+/K+-ATPases probably appeared late in evolution because they are restricted to animals, who evolved in the sea and were dependent on systems to extrude sodium. However, in plants and animals respectively, H+-ATPase and Na+lK+-ATPase serve analogous functions. Thus, in plants (and

Purification of heterologously expressed plant plasma membrane H+-ATPase by Ni2+-affinity chromatography

The ATP-driven plasma membrane proton pump of plants (PM-H+-ATPase) is a P-type ATPase that pumps protons out of the cytoplasm. The membrane potential and proton gradient generated are used to drive solute transport across the plasma membrane of plant cells. The study on structure-function relationships of this ATPase requires the availability of an effective system for expressing and isolating mutant forms of the ATPase and the ability to grow diffraction-quality crystals of the enzyme. To facilitate efficient purification of the Arabidopsis thaliana PM-H+-ATPase and its mutant proteins, we constructed a PM-H+-ATPase with an affinity tag of 6 histidine residues in its NH2-terminus (6H-AHA2). A (CAC)6-oligonucleotide cassette was inserted into a convenient EcoRV site, which coincided with the codons for Asp6 and Ile7 (FIG. I ) . The chimeric gene was placed under the control of the strong PMAl promoter and was subcloned into a yeast multicopy vector that was used to transform the yeast Saccharomyces cerevisiue. Production of 6H-AHA2 in the endoplasmic reticulum (ER) of transformed yeast cells was confirmed by SDS-PAGE (FIG. 2A) and Western blotting (FIG. 2B). Production was high and comparable to that of wild-type AHA2. After isolation of ER membranes from the transformed yeast cells, kinetic studies revealed the presence of ATPase activity with characteristics ( K r n , ~ ~ p = 1.2 mM; pH optimum = 6.6) comparable to those of the wild-type plant PM-H+-ATPase. These ER membranes were used for purification of the 6H-AHA2 polypeptide. ER proteins were solubilized with 0.3% (w/v) n-dodecyl P-D-maltoside (DDM) (detergent/ protein ratio = 3). Insoluble material was removed by centrifugation, and the supernatant was exposed to the Ni2+-nitrilotriacetic acid resin. The resin was packed into a column and washed with solutions containing DDM and soybean phosphatidylcholine and increasing concentrations of irnidazole. Purified PM-H+-ATPase could be detected in the fractions with 150 mM imidazole (FIG. 3). These fractions contained about 4% of the amount of ER protein and close to 27% of the ATPase-activity initially present in the ER. The presence of asolectin in the washing and elution solutions appeared essential. Without asolectin the PM-H+-