Vacuoles are multifunctional organelles essential for the sessile lifestyle of plants. Despite their central functions in cell growth, storage, and detoxification, knowledge about mechanisms underlying their biogenesis and associated protein trafficking pathways remains limited. Here, we show that in meristematic cells of the Arabidopsis thaliana root, biogenesis of vacuoles as well as the trafficking of sterols and of two major tonoplast proteins, the vacuolar H + -pyrophosphatase and the vacuolar H + -adenosinetriphosphatase, occurs independently of endoplasmic reticulum (ER)-Golgi and post-Golgi trafficking. Instead, both pumps are found in provacuoles that structurally resemble autophagosomes but are not formed by the core autophagy machinery. Taken together, our results suggest that vacuole biogenesis and trafficking of tonoplast proteins and lipids can occur directly from the ER independent of Golgi function.
The V-ATPase is a versatile proton-pump found in a range of endomembrane compartments yet the mechanisms governing its differential targeting remain to be determined. In Arabidopsis, VHA-a1 targets the V-ATPase to the TGN/EE whereas VHA-a2 and VHA-a3 are localized to the tonoplast. We report here that the VHA-a1 targeting domain serves as both an ER-exit and as a TGN/EE-retention motif and is conserved among seed plants. In contrast, Marchantia encodes a single VHA-isoform that localizes to the TGN/EE and the tonoplast in Arabidopsis. Analysis of CRISPR/Cas9 generated null alleles revealed that VHA-a1 has an essential function for male gametophyte development but acts redundantly with the tonoplast isoforms during vegetative growth. We propose that in the absence of VHA-a1, VHA-a3 is partially re-routed to the TGN/EE. Our findings contribute to understanding the evolutionary origin of V-ATPase targeting and provide a striking example that differential localization does not preclude functional redundancy.
AbstractVacuolar-type H+-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 Marchantia polymorpha 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.
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