Abstract: The retromer complex is a vital component of the endosomal protein sorting machinery necessary for sorting into both the endosome-to-Golgi retrieval pathway and also the endosome-to-cell-surface recycling pathway. Retromer mediates cargo selection through a trimeric complex comprising VPS35, VPS29 and VPS26, which is recruited to endosomes by binding to Rab7a and Snx3. Retromer function is linked to two distinct neurodegenerative diseases, Parkinson's disease and Alzheimer's disease and modulating retromer function has been proposed as an avenue to explore for a putative therapy in these conditions. We hypothesised that activating Rab7a to promote the recruitment of retromer to endosomes could positively modulate its activity. Here, we show that inhibition of the GTPase activating protein TBC1D5 can enhance Rab7a activation and lead to a gain of function for retromer.

Abstract: Retrograde vesicle trafficking pathways are responsible for returning membrane-associated components from endosomes to the Golgi apparatus and the endoplasmic reticulum (ER), and they are critical for maintaining organelle identity, lipid homeostasis, and many other cellular functions. The retrograde transport pathway has emerged as an important target for intravacuolar bacterial pathogens. The opportunistic pathogen Legionella pneumophila exploits both the secretory and recycling branches of the vesicle transport pathway for intracellular bacterial proliferation. Its Dot/Icm effector RidL inhibits the activity of the retromer by directly engaging retromer components. However, the mechanism underlying such inhibition remains unknown. Here we present the crystal structure of RidL in complex with VPS29, a subunit of the retromer. Our results demonstrate that RidL binds to a highly conserved hydrophobic pocket of VPS29. This interaction is critical for endosomal recruitment of RidL and for its inhibitory effects. RidL inhibits retromer activity by direct competition, in which it occupies the VPS29-binding site of the essential retromer regulator TBC1d5. The mechanism of retromer inhibition by RidL reveals a hotspot on VPS29 critical for recognition by its regulators that is also exploited by pathogens, and provides a structural basis for the development of small molecule inhibitors against the retromer.

Abstract: The large retromer complex participates in diverse endosomal trafficking pathways and is essential for plant developmental programs, including cell polarity, programmed cell death and shoot gravitropism in Arabidopsis. Here we demonstrate that an evolutionarily conserved VPS26 protein (VPS26C; At1G48550) functions in a complex with VPS35A and VPS29 necessary for root hair growth in Arabidopsis. Bimolecular fluorescence complementation showed that VPS26C forms a complex with VPS35A in the presence of VPS29, and this is supported by genetic studies showing that vps29 and vps35a mutants exhibit altered root hair growth. Genetic analysis also demonstrated an interaction between a VPS26C trafficking pathway and one involving the SNARE VTI13. Phylogenetic analysis indicates that VPS26C, with the notable exception of grasses, has been maintained in the genomes of most major plant clades since its evolution at the base of eukaryotes. To test the model that VPS26C orthologs in animal and plant species share a conserved function, we generated transgenic lines expressing GFP fused with the VPS26C human ortholog (HsDSCR3) in a vps26c background. These studies illustrate that GFP-HsDSCR3 is able to complement the vps26c root hair phenotype in Arabidopsis, indicating a deep conservation of cellular function for this large retromer subunit across plant and animal kingdoms.

Abstract: The polar transport of auxin controls many aspects of plant development. However, the molecular mechanisms underlying auxin tranport regulation remain to be further elucidated. We identified a mutant named as usl1 (unflattened and small leaves) in a genetic screen in Arabidopsis thaliana. The usl1 displayed multiple aspects of developmental defects in leaves, embryogenesis, cotyledons, silique phyllotaxy and lateral roots in addition to abnormal leaves. USL1 encodes a protein orthologous to the yeast vacuolar protein sorting (Vps) 38p and human UV RADIATION RESISTANCE‐ASSOCIATED GENE (UVRAG). Cell biology, Co‐IP/MS and yeast two‐hybrid were used to identify the function of USL1. USL1 colocalizes at the subcellular level with VPS29, a key factor of the retromer complex that controls auxin transport. The morphology of the VPS29‐associated late endosomes (LE) is altered from small dots in the wild‐type to aberrant enlarged circles in the usl1 mutants. The usl1 mutant synergistically interacts with vps29. We also found that USL1 forms a complex with AtVPS30 and AtVPS34. We propose that USL1 controls multiple aspects of plant development by affecting late endosome morphology and by regulating the PIN1 polarity. Our findings provide a new layer of the understanding on the mechanisms of plant development regulation.

Abstract: Endosomal traffic in the plant endomembrane system is a fundamental and complex process that controls many essential cellular, developmental, and physiological functions in plants, including cellular polarization, cytokinesis, metal ion homeostasis, pathogen defense, and hormone transport (1). The secretory and endocytic pathways represent two major anterograde protein transport routes for protein delivery into the vacuole in plant cells (Fig. 1 A ). In the secretory pathway, transportation of newly synthesized soluble vacuolar cargo proteins is mediated by the vacuolar sorting receptors (VSRs) (2). After delivery of the soluble cargos into an intermediate compartment, receptors are recycled by the attachment of conserved sorting nexins (SNXs) and the core subunits of retromer complex (VPS26, VPS29, and VPS35) to the membrane. Nevertheless, the precise localization of the SNXs and the retromer subunits, as well as the identity of the organelles from which VSRs are recycled, remains in debate (3, 4). During endocytosis, plasma membrane (PM) proteins are internalized and delivered into the trans -Golgi network (TGN)/early endosomes in plants (5). Ubiquitinated PM proteins are further sorted into the intralumenal vesicles of multivesicular bodies, previously identified as a prevacuolar compartment (6), by the endosomal sorting complex required for transport machinery for vacuolar degradation (7). Alternatively, PM proteins without a ubiquitin tag (or after removal of ubiquitin by a deubiquitinating enzyme) are recycled back to the PM from the TGN or recycling endosome (RE) (1, 8). In plants, numerous PM proteins undergo endocytosis and endosomal recycling, with the PIN-FORMED (PINs) transporters for the plant hormone auxin being the most studied (9). Polarized PM localization of PINs has a profound developmental importance and is tightly regulated by multiple endosomal trafficking routes, including endocytosis, endosomal recycling, and vacuolar degradation. PINs are internalized via clathrin-mediated endocytosis and then recycled back to the PM via the GNOM-positive putative RE … [↵][1]2To whom correspondence should be addressed. Email: ljiang{at}cuhk.edu.hk. [1]: #xref-corresp-1-1

Abstract: Coxiella burnetii is an intracellular bacterium that replicates within an expansive phagolysosome-like vacuole. Fusion between the Coxiella-containing vacuole (CCV) and late endosomes/multivesicular bodies requires Rab7, the HOPS tethering complex, and SNARE proteins, with actin also speculated to play a role. Here, we investigated the importance of actin in CCV fusion. Filamentous actin patches formed around the CCV membrane that were preferred sites of vesicular fusion. Accordingly, the mediators of endolysosomal fusion Rab7, VAMP7, and syntaxin 8 were concentrated in CCV actin patches. Generation of actin patches required C. burnetii type 4B secretion and host retromer function. Patches decorated with VPS29 and VPS35, components of the retromer, FAM21 and WASH, members of the WASH complex that engage the retromer, and Arp3, a component of the Arp2/3 complex that generates branched actin filaments. Depletion by siRNA of VPS35 or VPS29 reduced CCV actin patches and caused Rab7 to uniformly distribute in the CCV membrane. C. burnetii grew normally in VPS35 or VPS29 depleted cells, as well as WASH-knockout mouse embryo fibroblasts, where CCVs are devoid of actin patches. Endosome recycling to the plasma membrane and trans-Golgi of glucose transporter 1 (GLUT1) and cationic-independent mannose-6-phosphate receptor (CI-M6PR), respectively, was normal in infected cells. However, siRNA knockdown of retromer resulted in aberrant trafficking of GLUT1, but not CI-M6PR, suggesting canonical retrograde trafficking is unaffected by retromer disruption. Treatment with the specific Arp2/3 inhibitor CK-666 strongly inhibited CCV formation, an effect associated with altered endosomal trafficking of transferrin receptor. Collectively, our results show that CCV actin patches generated by retromer, WASH, and Arp2/3 are dispensable for CCV biogenesis and stability. However, Arp2/3-mediated production of actin filaments required for cargo transport within the endosomal system is required for CCV generation. These findings delineate which of the many actin related events that shape the endosomal compartment are important for CCV formation.

Abstract: The retromer cargo-selective complex (CSC) comprising Vps35, Vps29 and Vps26 mediates the endosome-to-Golgi retrieval of the cation-independent mannose 6-phosphate receptor (CIMPR). Or does it? Recently published data have questioned the validity of this long-established theory. Here, the evidence for and against a role for the retromer CSC in CIMPR endosome-to-Golgi retrieval is examined in the light of the new data that the SNX-BAR dimer is actually responsible for CIMPR retrieval.