A novel mechanism of P-type ATPase autoinhibition involving both termini of the protein

Abstract

The activity of many P-type ATPases is found to be regulated by interacting proteins or autoinhibitory elements located in N- or C-terminal extensions. An extended C terminus of fungal and plant P-type plasma membrane H(+)-ATPases has long been recognized to be part of a regulatory apparatus involving an autoinhibitory domain. Here we demonstrate that both the N and the C termini of the plant plasma membrane H(+)-ATPase are directly involved in controlling the pump activity state and that N-terminal displacements are coupled to secondary modifications taking place at the C-terminal end. This identifies the first group of P-type ATPases for which both ends of the polypeptide chain constitute regulatory domains, which together contribute to the autoinhibitory apparatus. This suggests an intricate mechanism of cis-regulation with both termini of the protein communicating to obtain the necessary control of the enzyme activity state.

FIGURE 1.

The plant PM H⁺-ATPase and the truncated PM H⁺-ATPases constructed in this study. A, schematic representation of the PM H⁺-ATPase, which is composed of discrete domains, namely a transmembrane domain (wheat), a phosphorylation domain (blue), a nucleotide-binding domain (red), and an actuator domain (yellow). The N terminus (N term) is located at the actuator domain. The regulatory C-terminal extension contains two autoinhibitory regions (I and II), and activation of the PM H⁺-ATPase occurs by phosphorylation (P) of Thr-947 and subsequent binding of 14-3-3 protein. The figure is based on the three-dimensional crystal structure of the plant PM H⁺-ATPase, where structural information was obtained for residues (Val-12 to Ile-844) but not for the regulatory C terminus (C term) or the extreme N terminus. B, wild-type and N-terminal truncated PM H⁺-ATPases used in this study. The N-terminal truncated H⁺-ATPase mutants are named according to the number of deleted amino acid residues.

FIGURE 2.

Modifications at the N terminus activate the plant PM H⁺-ATPase in a yeast complementation assay. A yeast complementation test was performed for no PM H⁺-ATPase, wild-type PM H⁺-ATPase (AHA2), C-terminal truncated PM H⁺-ATPase (aha2Δ73), N-terminal truncated PM H⁺-ATPase (ΔN10aha2), and a N-terminal truncated PM H⁺-ATPase with a His tag and a thrombin recognition sequence (His-Thrombin-ΔN10aha2). In the yeast strain RS-72, the endogenous yeast PM H⁺-ATPase, Pma1p, has been placed under the control of a galactose promoter, whereas the introduced, plasmid-borne plant PM H⁺-ATPases are under the control of the constitutive PMA1 promoter (24). Yeast growth on glucose is therefore dependent on a functional plasmid-borne PM H⁺-ATPase. Transformed yeast cells, RS-72, were spotted on either galactose-containing media (Gal) at pH 5.5 or glucose-containing media (Glu) at different pH values at two different concentrations (A₆₀₀ = 0.1 and A₆₀₀ = 0.01). Growth was recorded after 4 days at 30 °C. OD, optical density.

FIGURE 3.

Activation level of the PM H⁺-ATPase by truncation of the N terminus increases with the length of the deletion up to 10 amino acid residues and does not give an additive effect when combined with a C-terminal truncation. A, the yeast strain RS-72 transformed with N-terminal truncated mutants of either the wild-type plant PM H⁺-ATPase or the C-terminal truncated form of the enzyme was spotted on galactose- or glucose-containing media as in Fig. 2. OD, optical density. B, plasma membrane-enriched fractions of yeast strains expressing various N-terminal truncated mutants of the PM H⁺-ATPase were tested for expression level by immunostaining.

FIGURE 4.

The Δ N10aha2 PM H⁺-ATPase is activated biochemically. A, ATP dependence of ATP hydrolytic activity by wild-type PM H⁺-ATPase and the ΔN10aha2 PM H⁺-ATPase mutant at pH 7.0. B, pH dependence of ATP hydrolysis by wild-type PM H⁺-ATPase and the ΔN10aha2 PM H⁺-ATPase mutant. Purified plasma membrane-enriched fractions were analyzed in both A and B. □, wild-type AHA2; ■, ΔN10aha2. All experiments are represented as ± S.D.

FIGURE 5.

Molecular communication between the N and the C termini. Plasma membrane-enriched fractions of yeast strains expressing the various mutants of the PM H⁺-ATPase were run on SDS-PAGE and transferred to nitrocellulose for immunostaining or overlay assay. A, N-terminal truncated mutants of the PM H⁺-ATPase show increased level of phosphorylation and bind more 14-3-3 proteins than the wild type. pThr, phosphothreonine. B, the high capacity for 14-3-3 binding of the ΔN10aha2 PM H⁺-ATPase is strictly dependent on the penultimate Thr-947 residue.

FIGURE 6.

Activation of the PM H⁺-ATPase by N-terminal truncations is dependent on Thr-947 phosphorylation but can be surpassed by a modification of autoinhibitory region I. A, a yeast complementation test of aha2 T947A and N-terminal truncated mutants hereof. B, a yeast complementation test of aha2 G867A,T947A and N-terminal truncated mutants hereof. All yeast cells were spotted in optical densities (OD) of 0.1 and 0.01 on solid glucose-containing medium with different pH (5.5 and 4.5).

FIGURE 7.

Mutagenesis of the conserved Glu-10 residue in the N terminus of the PM H⁺-ATPase perturbs autoinhibition. RS-72 transformed with the indicated mutants was spotted on plates with different pH (5.5, 4.5, and 3.5) and in two different optical densities (OD), namely ODs of 0.1 and 0.01. Gly-867 resides in autoinhibitory region I, and Arg-913 resides in autoinhibitory region II (see Fig. 1A).