The Arabidopsis V-ATPase is localized to the TGN/EE via a seed plant-specific motif

Abstract

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.

Multiple functions of the vacuole in plant growth and fruit quality

“…How vacuoles originate remains a matter of debate, hence there is an urgent need to obtain experimental evidence supporting or opposing the various hypotheses that have been proposed. The hypothesis of direct initiation from the ER proposes that proteins on the tonoplast reach the vacuole from the ER, without passing through protein transport pathways (the Golgi apparatus) (Uemura & Ueda, 2014;Lupanga et al, 2020). Are there other quality control systems for such proteins?…”

Opposite polarity programs regulate asymmetric subsidiary cell divisions in grasses

“…The generation of the entry modules pGGA000, pGGB003, pGGC000, pGGD002, pGGE001, and pGGF000 are described in (Lampropoulos et al, 2013). The entry modules pGGD_linker-mVenus are described in (Waadt et al, 2020), and pGGZ004 is described in (Lupanga et al, 2020). The pGGB_3xNLS and pGGC_eGFP were generously provided by Karin Schumacher’s group.…”

CLCd and CLCf act redundantly at the TGN/EE and prevent acidification of the Golgi stack

“…CLCd has been reported to negatively regulate pathogen-triggered immunity in response to flg22 (Guo et al, 2014) whereas reduced V-ATPase activity dampens the flg22 response (Keinath et al, 2010) indicating that the functional relation between CLCs and the V-ATPase might be more complex and a more detailed characterisation of the single mutants might be required to reveal non-redundant functions. However, the fact that the combined loss of CLCd and CLCf causes male gametophyte lethality, a phenotype also observed in V-ATPase null mutants (Dettmer et al, 2005;Lupanga et al, 2020) also supports a role for CLC-activity in supporting acidification of the TGN/EE. Further analysis is required to determine if the underlying pollen defects are indeed similar, yet developing pollen is not the most suitable experimental system to study CLC-function in the TGN/EE in more detail.…”

“…The correct amiR-clcf cassette was verified by sequencing and subsequently used to generate the intermediate vector pGGN-6xOP:amiR-clcf via GG reaction with modules listed in Supplementary Table S2. Afterwards a final GG reaction was performed with pGGN-6xOP:amiR-clcf, pGGM-UBQ10:Lh4GR (Lupanga et al, 2020) and the destination vector pGGZ003 (Lampropoulos et al, 2013) to receive Dex:amiR-clcf.…”