In vivo cross-linking supports a head-to-tail mechanism for regulation of the plant plasma membrane P-type H+-ATPase

Abstract

In higher plants, a P-type proton-pumping ATPase generates the proton-motive force essential for the function of all other transporters and for proper growth and development. X-ray crystallographic studies of the plant plasma membrane proton pump have provided information on amino acids involved in ATP catalysis but provided no information on the structure of the C-terminal regulatory domain. Despite progress in elucidating enzymes involved in the signaling pathways that activate or inhibit this pump, the site of interaction of the C-terminal regulatory domain with the catalytic domains remains a mystery. Genetic studies have pointed to amino acids in various parts of the protein that may be involved, but direct chemical evidence for which ones are specifically interacting with the C terminus is lacking. In this study, we used in vivo cross-linking experiments with a photoreactive unnatural amino acid, p-benzoylphenylalanine, and tandem MS to obtain direct evidence that the C-terminal regulatory domain interacts with amino acids located within the N-terminal actuator domain. Our observations are consistent with a mechanism in which intermolecular, rather than intramolecular, interactions are involved. Our model invokes a "head-to-tail" organization of ATPase monomers in which the C-terminal domain of one ATPase molecule interacts with the actuator domain of another ATPase molecule. This model serves to explain why cross-linked peptides are found only in dimers and trimers, and it is consistent with prior studies suggesting that within the membrane the protein can be organized as homopolymers, including dimers, trimers, and hexamers.

Keywords: Arabidopsis thaliana; H+-ATPase; mass spectrometry (MS); protein cross-linking; protein-protein interaction.

Figure 1.

Two-dimensional structure of AHA2 with BPA incorporated at different positions (indicated with purple stars) in AHA2, including the actuator domain (orange), phosphorylation domain (blue), nucleotide-binding domain (red), and the regulatory domain (last 105 amino acids at the C terminus) that includes the three regulatory regions R-I (black), R-B (white), and R-II (black).

Figure 2.

BPA was incorporated at different positions of AHA2 and cross-linked in vivo with UV irradiation. A, whole-cell extract from yeast cells expressing His₆-HA–tagged AHA2 amber mutants and the tRNA synthetase/tRNASup in the absence (−) or presence of 2 mm BPA(+) were analyzed by Western blotting with anti-HA antibody. B, drop test for the growth of yeast strains expressing BPA–AHA2s on plates containing either 2% galactose or 2% glucose + 2 mm BPA at pH ∼6.5. Growth was recorded after 4 days. C, cells expressing BPA-containing AHA2 were kept in the dark (−) or exposed to UV 365 nm (+), and AHA2 was purified from cell extracts using nickel-Sepharose resin, followed by Western blotting with anti-HA antibody. The cross-linked species are observed as higher molecular bands in the upper part of the gel above the monomeric 110-kDa ATPase polypeptide. D, Strep-HA–tagged AHA2s containing BPA was purified with StrepTactin-Sepharose (GE Healthcare) resin and analyzed by NuPAGE Tris acetate gel. On this gel, the cross-link bands are indicated as a dimer and a trimer of AHA2.

Figure 3.

In UV-irradiated samples, MS and MS/MS analysis show a cross-linked product between S229BPA and peptide ⁸⁸⁹EAVNIFPEKGSYR⁹⁰¹ in the trimer and dimer bands but not in the monomer band of AHA2-S229BPA. A, extracted ion chromatogram and mass spectrum showing a quadruply charged ion at m/z 734.109 in the trimer and dimer bands. B and C, fragment ion mass spectrum (MS/MS) of the precursor at m/z 734.109 in the trimer band (B) and dimer band (C). The fragment ions from peptide α are in red and from peptide β are in blue.

Figure 4.

MS and MS/MS analysis show a cross-linked product between H225BPA and peptide ⁸⁸⁹EAVNIFPEK⁸⁹⁷ in the trimer and dimer bands but not in the monomer band of AHA2-H225BPA upon UV irradiation. A, extracted ion chromatogram and mass spectrum showing a quadruply charged ion at m/z 728.621 in the trimer and dimer bands. B and C, fragment ion mass spectrum (MS/MS) of the precursor at m/z 728.621 in the trimer band (B) and dimer band (C). The fragment ions from peptide α are in red and from peptide β are in blue.

Figure 5.

Model describing a head-to-tail interaction of the N-terminal actuator domain and the C-terminal regulatory domain of the 100,000 Da H⁺-ATPase in oligomeric AHA2. In the active state, the protein can exist as a dimer, as a trimer, or as a higher order oligomer in which the C-terminal domain of one monomer interacts with and inhibits the actuator movement of the neighboring unit. Under normal conditions, this inhibition may be switched on or off as the dynamic C-terminal domains swing back and forth to the N-terminal actuator domains. The enzyme can also enter a stable hyperactive state, as shown in the hexamers on the right, in which phosphorylation of the penultimate threonine results in the binding of a 14-3-3 protein to the C-terminal domain of each monomer, locking it into place so that it can no longer swing back to bind the actuator domain. It remains unclear whether the interaction between the actuator domain and the regulatory domain specifically mediates oligomerization, although hexamer formation may be required for hyperactivation.