Retromer Subunits VPS35A and VPS29 Mediate Prevacuolar Compartment (PVC) Function in Arabidopsis

Abstract

Intracellular protein routing is mediated by vesicular transport which is tightly regulated in eukaryotes. The protein and lipid homeostasis depends on coordinated delivery of de novo synthesized or recycled cargoes to the plasma membrane by exocytosis and their subsequent removal by rerouting them for recycling or degradation. Here, we report the characterization of protein affected trafficking 3 (pat3) mutant that we identified by an epifluorescence-based forward genetic screen for mutants defective in subcellular distribution of Arabidopsis auxin transporter PIN1-GFP. While pat3 displays largely normal plant morphology and development in nutrient-rich conditions, it shows strong ectopic intracellular accumulations of different plasma membrane cargoes in structures that resemble prevacuolar compartments (PVC) with an aberrant morphology. Genetic mapping revealed that pat3 is defective in vacuolar protein sorting 35A (VPS35A), a putative subunit of the retromer complex that mediates retrograde trafficking between the PVC and trans-Golgi network. Similarly, a mutant defective in another retromer subunit, vps29, shows comparable subcellular defects in PVC morphology and protein accumulation. Thus, our data provide evidence that the retromer components VPS35A and VPS29 are essential for normal PVC morphology and normal trafficking of plasma membrane proteins in plants. In addition, we show that, out of the three VPS35 retromer subunits present in Arabidopsis thaliana genome, the VPS35 homolog A plays a prevailing role in trafficking to the lytic vacuole, presenting another level of complexity in the retromer-dependent vacuolar sorting.

Retromer Contributes to Immunity-Associated Cell Death in Arabidopsis

“…Plant retromer functions in endosomal protein sorting and vacuolar trafficking during development but has not been implicated in immunity before (Reyes et al, 2011;Robinson et al, 2012;Nodzynski et al, 2013). We demonstrate that VPS35-dependent trafficking pathways contribute to TIR-NB-LRR and CC-NB-LRR protein-mediated autoimmunity and HR cell death.…”

“…These subunits localize to the prevacuolar compartment (PVC) and interact with each other (Oliviusson et al, 2006;Jaillais et al, 2007;Zelazny et al, 2013). However, in contrast with other VPS35 homologs, the subcellular localization and interaction capacity of VPS35B have not been studied in detail (Nodzynski et al, 2013;Zelazny et al, 2013). Therefore, we generated transgenic lines expressing a native promoter-driven VPS35B-GREEN FLUORESCENT PROTEIN (GFP) fusion in vps35b-2 and subsequently introduced subcellular compartment markers for colocalization studies.…”

“…Retromer is implicated in endomembrane trafficking and cargo delivery to the lytic vacuole, which plays essential roles in HR execution (Hara-Nishimura and Hatsugai, 2011;Robinson et al, 2012;Nodzynski et al, 2013). Thus, we investigated whether HR suppression in retromer mutants might be linked to alterations in the morphology and function of endosomal and vacuolar compartments.…”

The dynamics of plant plasma membrane proteins: PINs and beyond

“…SNX1 has also been implicated in the recycling of internalized IRT1 from late endosomes to prevent its premature degradation (Ivanov et al, 2014). The VPS35A retromer subunit, by contrast, appears to be required for the functionality of late endosomal compartments, reflecting the differing functions of distinct elements of the plant retromer machinery (Nodzynski et al, 2013).…”

Analysis of the role of RNS2 and autophagy in the turnover of ribosomal RNA in Arabidopsis thaliana

“…Retromers are involved in transporting organelles after they depart the Golgi and trafficking proteins to the lysosome or vacuole (Robinson et al 2012). Retromers are also important for plant development and mediate prevacuolar compartment formation as well as endosomal trafficking (Reyes et al 2011;Nodzynski et al 2013). During the hypersensitive response to perceived pathogens in plants lacking retromers, autophagy and other vacuolar lytic processes needed for the programmed cell death hypersensitive response are impaired, implicating retromers in proper vacuolar-and autophagy-mediated cell death (Munch et al 2015).…”