Inter- and Intra-Molecular Interactions of The Arabidopsis Plasma Membrane Proton Pump Revealed Using a Mass Spectrometric-Cleavable Crosslinker

ABSTRACT: In plants and fungi, the plasma membrane proton pump (H+-ATPase) establishes an electrochemical gradient across the plasma membrane which serves as the driving force for the secondary transport of ions and nutrients across the cell membrane. This is an essential enzyme that functions in many important processes including stomatal movement, cell elongation, and cellular responses to stimuli from hormones, light, and other environmental conditions. Therefore, understanding how the activity of the H+-ATPase is regulated is important to understand how plants adapt to different growth conditions. The autoinhibitory effect of the C- terminal regulatory domain of H+-ATPase is well established and is thought to be mediated by interactions with the catalytic domains. Here, using the lysine reactive mass spectrometry cleavable crosslinker DSSO, we found that the C-terminal domain of the Arabidopsis H+- ATPase 2 (AHA2) crosslinked extensively with the actuator, nucleotide-binding, and phosphorylation domains, suggesting that the C-terminal domain regulates the catalytic cycle by modulating the relative positions of these domains. Interestingly, several C-terminal crosslinks occurred near a predicted proton binding site (Asp-684 in TM6), suggesting that the C-terminal domain may regulate proton efflux. Additionally, crosslinks between the C-terminal domain and other domains of AHA2 were detected in a monomeric protein resolved on SDS-PAGE, suggesting that intramolecular interactions may also be involved in the regulation of enzyme activity. Finally, we observed mixed-isotope crosslinking between unlabeled (14N-AHA2) and labeled (15N-AHA2) between the C-terminal domain and other domains of AHA2, supporting our model that oligomeric H+-ATPase may autoinhibit the neighboring monomer in a “head to tail” configuration.

INSTRUMENT(S): Orbitrap Fusion Lumos

ORGANISM(S): Saccharomyces Cerevisiae (baker's Yeast) Arabidopsis Thaliana (mouse-ear Cress)

SUBMITTER: Thao Thi Nguyen  

LAB HEAD: Michael R. Sussman

PROVIDER: PXD018219 | Pride | 2023-06-01

REPOSITORIES: Pride