Abstract
The Arabidopsis plasma membrane proton P‐type ATPases AHA1 and AHA2 are responsible for generating a proton motive force across the plasma membrane and are essential for successful embryo development. We report the translational fusion of AHA1 to a tandem affinity purification tag and stable expression of the tagged protein in transgenic double aha1/aha2 knockout plants. TAP tagged AHA1 complements the double knockout lethality, Western blots show the protein is targeted to the plasma membrane, and plants grow normally under standard conditions. An affinity purification mass spectrometry (APMS) workflow was used to identify proteins that co‐purify with TAP tagged AHA1 from plasma membrane enriched fractions. We identified two proteins co‐enriched with AHA1 by label free MS and an additional 17 using a metabolically labeled approach. Several of the proteins identified are consistent with previous literature on AHA1 protein interactions, while others are novel. In conclusion, we have generated transgenic Arabidopsis lines in which a TAP tagged AHA1 has complemented all endogenous AHA1 and AHA2 function and have shown that these plants can be used to specifically purify AHA1 and identify in planta interacting proteins by mass spectrometry.
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