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

Thao T Nguyen,Grzegorz Sabat,Michael R Sussman

doi:10.1074/jbc.ra118.003528

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.

Highlights

The plasma membrane proton pump (Hϩ-ATPase) is the primary energy-transducing transporter in fungi and plants
We exploited amber codon recoding to site- incorporate the unnatural photolabile amino acid BPA, which can cross-link to alkyl carbons of nearby amino acid residues [31], into His6–HA-tagged AHA2 using the S. cerevisiae RS72 strain as the expression system [32]
We present the first evidence that the C-terminal domain of AHA2 directly interacts with the N-terminal actuator domain in vivo by using an unnatural amino acid as a photoaffinity cross-linker and light delivered to the enzyme in intact cells

Summary

Results

Genetic truncation and proteolytic experiments indicate that the C-terminal domain inhibits Hϩ-ATPase catalytic function, the exact location where it interacts remains a mystery. We initially attempted to incorporate BPA directly into the very end of the protein within the C terminus, but we obtained a very low yield of BPA–AHA2 at these sites, major products were truncated proteins (cf., Fig. 2A and Table S1). To circumvent this problem, we placed BPA at several dozen locations scattered throughout the protein (Fig. 1), especially within the N-terminal actuator domain that was predicted to be involved in interaction with the C-terminal domain in previous genetic studies [8, 9]. As expected, when BPA was not present in the culture medium, very few or no full-length proteins were detected, whereas upon addition of BPA, full-length

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Discussion

Experimental procedures