Investigating the function and regulation of the Arabidopsis plasma membrane proton pump AHA1 using reverse genetics and mass spectrometry

Plasma membrane proton pumps (H+-ATPases) are essential integral membrane proteins found in yeast and plants. These proteins use up to one-third of the cellular ATP pool to establish an electrochemical proton gradient across the membrane, which is essential for driving diverse downstream functions. The in planta roles of the most abundant Arabidopsis PM H+-ATPases, AHA1 and AHA2, remain unclear due to the lack of strong phenotypes in single mutants and the embryo lethal phenotype of the double mutants. Heterologous expression in yeast has provided a simplified system for studying plant H+-ATPases, but these experiments provide limited basis for understanding the native physiological roles of these proteins. Herein, I describe experiments aimed at understanding the function and regulation of AHA1 using reverse genetics and mass spectrometry based approaches. Post-translational protein phosphorylation is a common mechanism of protein regulation, and multiple phosphorylated residues have been identified in AHA proteins. Phosphorylated residues are especially abundant in the AHA C-terminal regulatory domain. Using phospho-mimic (Asp) and non-phosphorylateable (Ala) point mutant transgenes, I investigated the requirement of post-translational phosphorylation for essential AHA1 function at two C-terminal residues. While the penultimate residue, Thr948, was strictly essential for protein function, mutations at Ser904 were well tolerated in planta. AHA proteins are hypothesized to play roles in important physiological processes and are highly regulated enzymes. Although one would predict that protein interactions (for example, with kinases and phosphatases) are necessary to mediate these diverse functions, only a few interactions have been identified to date. Affinity purification combined with analysis of purified proteins by biochemical methods including mass spectrometry is one way to identify protein interactions. In order to facilitate affinity purification of AHA1 from plant tissue, I translationally fused a tandem affinity purification tag to an AHA1 genomic clone. I then established the ability of this transgene to complement aha1/aha2 embryo lethality and subsequently used an affinity purification mass spectrometry approach to identify AHA1 co-purifying proteins.