The Arabidopsis V-ATPase is localized to the TGN/EE via a seed plant specific motif and acts in a partially redundant manner with the tonoplast enzyme

Abstract

<jats:title>Abstract</jats:title><jats:p>Vacuolar-type H<jats:sup>+</jats:sup>-ATPases (V-ATPases) are versatile proton pumps that control the pH of many intracellular compartments in all eukaryotic cells. The localization of the Arabidopsis V-ATPase was previously shown to be determined by the isoforms of subunit a (VHA-a). The incorporation of VHA-a1 targets the V-ATPase to the trans-Golgi network/early endosome (TGN/EE) whilst the incorporation of VHA-a2 or VHA-a3 targets the V-ATPase to the tonoplast. By employing chimeric proteins and site directed mutagenesis we identified a targeting domain (a1-TD) containing an acidic cluster in the N-terminus of VHA-a1 that serves as both an ER export signal and as a TGN retention motif. The a1-TD is conserved among seed plants and we confirmed experimentally that its presence is predictive of TGN/EE- localization. In contrast to many other non-seed plants, the liverwort <jats:italic>Marchantia polymorpha</jats:italic> encodes only a single VHA-a subunit (MpVHA-a) and we show here that it is predominantly localized at the tonoplast. In our attempts to determine if MpVHA-a can functionally replace the Arabidopsis VHA-a isoforms, we used CRISPR/Cas9 to generate null-alleles lacking VHA-a1 and discovered that its function is essential for male gametophyte development but can be replaced by VHA-a2 and VHA-a3 during vegetative growth.</jats:p>