ORCID IDs: 0000-0003-2780-4888 (C.R.); 0000-0002-8800-2400 (V.R.)Prior to the release of their cargoes into the vacuolar lumen, sorting endosomes mature into multivesicular bodies (MVBs) through the action of ENDOSOMAL COMPLEX REQUIRED FOR TRANSPORT (ESCRT) protein complexes. MVB-mediated sorting of high-affinity phosphate transporters (PHT1) to the vacuole limits their plasma membrane levels under phosphatesufficient conditions, a process that allows plants to maintain phosphate homeostasis. Here, we describe ALIX, a cytosolic protein that associates with MVB by interacting with ESCRT-III subunit SNF7 and mediates PHT1;1 trafficking to the vacuole in Arabidopsis thaliana. We show that the partial loss-of-function mutant alix-1 displays reduced vacuolar degradation of PHT1;1. ALIX derivatives containing the alix-1 mutation showed reduced interaction with SNF7, providing a simple molecular explanation for impaired cargo trafficking in alix-1 mutants. In fact, the alix-1 mutation also hampered vacuolar sorting of the brassinosteroid receptor BRI1. We also show that alix-1 displays altered vacuole morphogenesis, implying a new role for ALIX proteins in vacuolar biogenesis, likely acting as part of ESCRT-III complexes. In line with a presumed broad target spectrum, the alix-1 mutation is pleiotropic, leading to reduced plant growth and late flowering, with stronger alix mutations being lethal, indicating that ALIX participates in diverse processes in plants essential for their life. INTRODUCTIONTrafficking of cargo proteins coming from the plasma membrane (PM) or the Golgi apparatus (GA) to the vacuole occurs through multivesicular bodies (MVBs) (Winter and Hauser, 2006). These organelles, also termed late endosomes or prevacuolar compartments (PVCs), contain internal vesicles that will be delivered, together with their cargoes, into the lumen of vacuoles/lysosomes upon MVB fusion with the tonoplast (Winter and Hauser, 2006). This process plays a central role in controlling the reutilization, storage, or degradation of membrane components and thus regulates fundamental biological processes including membrane turnover, defense against pathogens, development, hormone transport, nutrient uptake, and cell signaling. In the case of membrane-associated regulatory proteins, the MVB route allows modulation of their function by regulating their abundance at the PM and in other vesicular compartments (e.g., endosomes).The sorting of most integral membrane proteins into intraluminal vesicles (ILVs) is dependent on the attachment of ubiquitin to their cytosolic domains, although ubiquitin-independent sorting mechanisms also exist (McNatt et al., 2007). Selective packaging of protein cargoes into ILVs of MVB is mediated by ESCRT (ENDOSOMAL SORTING COMPLEXES REQUIRED FOR TRANSPORT) protein complexes (Conibear, 2002;Winter and Hauser, 2006;Nickerson et al., 2007;Henne et al., 2011). The latter consist of several cytosolic proteins of the VPS-E (class E Vacuolar Protein Sorting) class that are organized into five complexes: ESCRT-0...

Ubiquitination is a signal for various cellular processes, including for endocytic degradation of plasma membrane cargos. Ubiquitinating as well as deubiquitinating enzymes (DUBs) can regulate these processes by modifying the ubiquitination status of target protein. Although accumulating evidence points to the important regulatory role of DUBs, the molecular basis of their regulation is still not well understood. Associated molecule with the SH3 domain of signal transduction adaptor molecule (STAM) (AMSH) is a conserved metalloprotease DUB in eukaryotes. AMSH proteins interact with components of the endosomal sorting complex required for transport (ESCRT) and are implicated in intracellular trafficking. To investigate how the function of AMSH is regulated at the cellular level, we carried out an interaction screen for the Arabidopsis AMSH proteins and identified the Arabidopsis homolog of apoptosis-linked gene-2 interacting protein X (ALIX) as a protein interacting with AMSH3 in vitro and in vivo. Analysis of alix knockout mutants in Arabidopsis showed that ALIX is essential for plant growth and development and that ALIX is important for the biogenesis of the vacuole and multivesicular bodies (MVBs). Cell biological analysis revealed that ALIX and AMSH3 colocalize on late endosomes. Although ALIX did not stimulate AMSH3 activity in vitro, in the absence of ALIX, AMSH3 localization on endosomes was abolished. Taken together, our data indicate that ALIX could function as an important regulator for AMSH3 function at the late endosomes.Arabidopsis | intracellular trafficking | ubiquitin |

Phosphate (Pi) is a macronutrient that is essential for plant life. Several regulatory components involved in Pi homeostasis have been identified, revealing a very high complexity at the cellular and subcellular levels. Determining the Pi content in plants is crucial to understanding this regulation, and short real-time(33)Pi uptake imaging experiments have shown Pi movement to be highly dynamic. Furthermore, gene modulation by Pi is finely controlled by localization of this ion at the tissue as well as the cellular and subcellular levels. Deciphering these regulations requires access to and quantification of the Pi pool in the various plant compartments. This review presents the different techniques available to measure, visualize and trace Pi in plants, with a discussion of the future prospects.