Mechanisms governing the endosomal membrane recruitment of the core retromer in Arabidopsis.

The retromer complex localizes to endosomal membranes and is involved in protein trafficking. In mammals, it is composed of a dimer of sorting nexins and of the core retromer consisting of vacuolar protein sorting (VPS)26, VPS29, and VPS35. Although homologs of these proteins have been identified in plants, how the plant retromer functions remains elusive. To better understand the role of VPS components in the assembly and function of the core retromer, we characterize here Arabidopsis vps26-null mutants. We show that impaired VPS26 function has a dramatic effect on VPS35 levels and causes severe phenotypic defects similar to those observed in vps29-null mutants. This implies that functions of plant VPS26, VPS29, and VPS35 are tightly linked. Then, by combining live-cell imaging with immunochemical and genetic approaches, we report that VPS35 alone is able to bind to endosomal membranes and plays an essential role in VPS26 and VPS29 membrane recruitment. We also show that the Arabidopsis Rab7 homolog RABG3f participates in the recruitment of the core retromer to the endosomal membrane by interacting with VPS35. Altogether our data provide original information on the molecular interactions that mediate assembly of the core retromer in plants.

Analyses of sorting nexins reveal distinct retromer-subcomplex functions in development and protein sorting in Arabidopsis thaliana.

Sorting nexins (SNXs) are conserved eukaryotic proteins that associate with three types of vacuolar protein sorting (VPS) proteins to form the retromer complex. How SNXs act in this complex and whether they might work independently of the retromer remains elusive. Here, we show by genetic and cell imaging approaches that the Arabidopsis thaliana SNX1 protein recruits SNX2 at the endosomal membrane, a process required for SNX1-SNX2 dimer activity. We report that, in contrast with the mammalian retromer, SNXs are dispensable for membrane binding and function of the retromer complex. We also show that VPS retromer components can work with or independently of SNXs in the trafficking of seed storage proteins, which reveals distinct functions for subcomplexes of the plant retromer. Finally, we provide compelling evidence that the combined loss of function of SNXs and VPS29 leads to embryo or seedling lethality, underlining the essential role of these proteins in development.

AtSNX1 defines an endosome for auxin-carrier trafficking in Arabidopsis.

Polarized cellular distribution of the phytohormone auxin and its carriers is essential for normal plant growth and development. Polar auxin transport is maintained by a network of auxin influx (AUX) and efflux (PIN) carriers. Both auxin transport and PIN protein cycling between the plasma membrane and endosomes require the activity of the endosomal GNOM; however, intracellular routes taken by these carriers remain largely unknown. Here we show that Arabidopsis thaliana SORTING NEXIN 1 (AtSNX1) is involved in the auxin pathway and that PIN2, but not PIN1 or AUX1, is transported through AtSNX1-containing endosomes. We demonstrate that the snx1-null mutant exhibits multiple auxin-related defects and that loss of function of AtSNX1 severely enhances the phenotype of a weak gnom mutant. In root cells, we further show that AtSNX1 localizes to an endosomal compartment distinct from GNOM-containing endosomes, and that PIN2 accumulates in this compartment after treatment with the phosphatidylinositol-3-OH kinase inhibitor wortmannin or after a gravity stimulus. Our data reveal the existence of a novel endosomal compartment involved in PIN2 endocytic sorting and plant development.

Cell shape: A ROP regulatory tug-of-war in pavement cell morphogenesis.

ROP GTPases coordinate the complex morphogenesis of leaf pavement cells. Four new studies reveal how ROP activity is regulated by both activating and inhibitory proteins to orchestrate cell lobe formation in response to local extracellular cues.

Developmental control of plant Rho GTPase nano-organization by the lipid phosphatidylserine.

Rho guanosine triphosphatases (GTPases) are master regulators of cell signaling, but how they are regulated depending on the cellular context is unclear. We found that the phospholipid phosphatidylserine acts as a developmentally controlled lipid rheostat that tunes Rho GTPase signaling in Arabidopsis Live superresolution single-molecule imaging revealed that the protein Rho of Plants 6 (ROP6) is stabilized by phosphatidylserine into plasma membrane nanodomains, which are required for auxin signaling. Our experiments also revealed that the plasma membrane phosphatidylserine content varies during plant root development and that the level of phosphatidylserine modulates the quantity of ROP6 nanoclusters induced by auxin and hence downstream signaling, including regulation of endocytosis and gravitropism. Our work shows that variations in phosphatidylserine levels are a physiological process that may be leveraged to regulate small GTPase signaling during development.

Evidence for a sorting endosome in Arabidopsis root cells.

In eukaryotic cells, the endocytic and secretory pathways are key players in several physiological processes. These pathways are largely inter-connected in animal and yeast cells through organelles named sorting endosomes. Sorting endosomes are multi-vesicular compartments that redirect proteins towards various destinations, such as the lysosomes or vacuoles for degradation, the trans-Golgi network for retrograde transport and the plasma membrane for recycling. In contrast, cross-talk between the endocytic and secretory pathways has not been clearly established in plants, especially in terms of cargo protein trafficking. Here we show by co-localization analyses that endosomes labelled with the AtSORTING NEXIN1 (AtSNX1) protein overlap with the pre-vacuolar compartment in Arabidopsis root cells. In addition, alteration of the routing functions of AtSNX1 endosomes by drug treatments leads to mis-routing of endocytic and secretory cargo proteins. Based on these results, we propose that the AtSNX1 endosomal compartment represents a sorting endosome in root cells, and that this specialized organelle is conserved throughout eukaryotes.

The retromer protein VPS29 links cell polarity and organ initiation in plants.

A key feature of plants (as opposed to animals) is their ability to establish new organs not only during embryogenesis, but also throughout their development. A master regulator of organ initiation in plants is the phytohormone auxin. Auxin acts locally as a morphogen and is directionally transported from cell to cell by polarized auxin efflux carriers, termed PIN-FORMED (PIN) proteins. Here we report that the Arabidopsis ortholog of the yeast and mammalian vacuolar protein sorting 29 (VPS29), a member of the retromer complex, mediates the formation of new axes of development. Furthermore, we show that VPS29 is required for endosome homeostasis, PIN protein cycling, and dynamic PIN1 repolarization during development. We propose a model that links VPS29 function, PIN1 polarity, and organ initiation in plants.

Refolding from denatured inclusion bodies, purification to homogeneity and simplified assay of MGDG synthases from land plants.

In plant cells, the synthesis of monogalactosyldiacylglycerol (MGDG) is catalyzed within plastid envelope membranes by MGD proteins. MGDG synthesis was also reported in apicomplexan parasites, a phylum of protists harbouring a plastid that proved essential for the parasite survival. MGD activity is therefore a potent target for herbicidal and anti-parasitic molecules. In this study, we describe a detailed in vitro refolding protocol for denatured recombinant MGD accumulated in inclusion bodies from transformed Escherichia coli. The refolding process was dependent on CHAPS detergent and lipids, such as diacylglycerol and phosphatidylglycerol, as well as bivalent metals. Owing to this refolding procedure, the recombinant MGD protein from spinach was purified to homogeneity, allowing a definite characterization of its non-processivity and an investigation of its dimerization using cross-linking reagents. Additionally, using the portion of recombinant enzyme that accumulates in an active form in bacterial membranes, we developed a miniature assay for high-throughput screening for inhibitors.