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.     

Retromer association with membranes: Plants have their own rules!

The retromer is an endosome-localized complex involved in protein trafficking. To better understand its function and regulation in plants, we recently investigated how Arabidopsis retromer subunits assemble and are targeted to endosomal membranes and highlighted original features compared with mammals. We characterized Arabidopsis vps26 null mutant and showed that it displays severe developmental defaults similar to those observed in vps29 mutant. Here, we go further by describing new phenotypic defects associated with loss of VPS26 function, such as inhibition of lateral root initiation. Recently, we showed that VPS35 subunit plays a crucial role in the recruitment of the plant retromer to endosomes, probably through an interaction with the Rab7 homolog RABG3f. In this work, we now show that contrary to mammals, Arabidopsis Rab5 homologs do not seem to be necessary for the recruitment of the core retromer to endosomal membranes, which highlights a new specificity of the plant retromer.     

Arabidopsis VPS35, a retromer component, is required for vacuolar protein sorting and involved in plant growth and leaf senescence

The retromer complex is responsible for retrograde transport,which is coordinated with anterograde transport in the secretorypathway including vacuolar protein sorting. Yeast VPS35 is acomponent of the retromer complex that is essential for recognitionof specific cargo molecules. The physiological function of VPS35has not been determined in vacuolar protein sorting in higherorganisms. Arabidopsis thaliana has three VPS35 homologs designatedVPS35a, VPS35b and VPS35c. We isolated four vps35 mutants (vps35a-1,vps35b-1, vps35b-2 and vps35c-1) and then generated four doublemutants and one triple mutant. vps35a-1 vps35c-1 exhibited nounusual phenotypes. On the other hand, vps35b-1 vps35c-1 andthe triple mutant (vps35a-1 vps35b-2 vps35c-1) exhibited severephenotypes: dwarfism, early leaf senescence and fragmentationof protein storage vacuoles (PSVs). In addition, these mutantsmis-sorted storage proteins by secreting them out of the cellsand accumulated a higher level of vacuolar sorting receptor(VSR) than the wild type. VPS35 was localized in pre-vacuolarcompartments (PVCs), some of which contained VSR. VPS35 wasimmunoprecipitated with VPS29/MAG1, another component of theretromer complex. Our findings suggest that VPS35, mainly VPS35b,is involved in sorting proteins to PSVs in seeds, possibly byrecycling VSR from PVCs to the Golgi complex, and is also involvedin plant growth and senescence in vegetative organs.     

Assembly and Solution Structure of the Core Retromer Protein Complex

Retromer is a peripheral membrane protein complex that has pleiotropic roles in endosomal membrane trafficking. The core of retromer possesses three subunits, VPS35, VPS29 and VPS26, that play different roles in binding to cargo, regulatory proteins and complex stabilization. We have performed an investigation of the thermodynamics of core retromer assembly using isothermal titration calorimetry (ITC) demonstrating that VPS35 acts as the central subunit to which VPS29 and VPS26 bind independently. Furthermore, we confirm that the conserved PRLYL motif of the large VPS35 subunit is critical for direct VPS26 interaction. Heat capacity measurements of VPS29 and VPS26 binding to VPS35 indicate extensive binding interfaces and suggest conformational alterations in VPS29 or VPS35 upon complex formation. Solution studies of the retromer core using small‐angle X‐ray scattering allow us to propose a model whereby VPS35 forms an extended platform with VPS29 and VPS26 bound at distal ends, with the potential for forming dimeric assemblies.     

Identification of a conserved motif required for Vps35p/Vps26p interaction and assembly of the retromer complex

The retromer complex is a conserved cytoplasmic coat complex that mediates the endosome-to-Golgi retrieval of vacuole/lysosome hydrolase receptors in yeast and mammals. The recognition of cargo proteins by the retromer is performed by the Vps35p/VPS35 (where Vps is vacuolar protein sorting) component, which together with Vps26p/VPS26 and Vps29p/VPS29, forms the cargo-selective subcomplex. In this report, we have identified a highly-conserved region of Vps35p/VPS35 that is essential for the interaction with Vps26p/VPS26 and for assembly of the retromer complex. Mutation of residues within the conserved region results in Vps35p/VPS35 mutants, which cannot bind to Vps26p/VPS26 and are not efficiently targeted to the endosomal membrane. These data implicate Vps26p/VPS26 in regulating Vps35p/VPS35 membrane association and therefore suggest a role for Vps26p/VPS26 in cargo recognition.     

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.     

VPS18-regulated vesicle trafficking controls the secretion of pectin and its modifying enzyme during pollen tube growth in Arabidopsis

In eukaryotes, homotypic fusion and vacuolar protein sorting (HOPS) as well as class C core vacuole/endosome tethering (CORVET) are evolutionarily conserved membrane tethering complexes that play important roles in lysosomal/vacuolar trafficking. Whether HOPS and CORVET control endomembrane trafficking in pollen tubes, the fastest growing plant cells, remains largely elusive. In this study, we demonstrate that the four core components shared by the two complexes, Vacuole protein sorting 11 (VPS11), VPS16, VPS33, and VPS18, are all essential for pollen tube growth in Arabidopsis thaliana and thus for plant reproduction success. We used VPS18 as a representative core component of the complexes to show that the protein is localized to both multivesicular bodies (MVBs) and the tonoplast in a growing pollen tube. Mutant vps18 pollen tubes grew more slowly in vivo, resulting in a significant reduction in male transmission efficiency. Additional studies revealed that membrane fusion from MVBs to vacuoles is severely compromised in vps18 pollen tubes, corroborating the function of VPS18 in late endocytic trafficking. Furthermore, vps18 pollen tubes produce excessive exocytic vesicles at the apical zone and excessive amounts of pectin and pectin methylesterases in the cell wall. In conclusion, this study establishes an additional conserved role of HOPS/CORVET in homotypic membrane fusion during vacuole biogenesis in pollen tubes and reveals a feedback regulation of HOPS/CORVET in the secretion of cell wall modification enzymes of rapidly growing plant cells.

Arabidopsis VPS18 plays an important role in regulating pollen tube growth through mediating the late endocytic trafficking and secretion of pectin and associated enzymes to the cell wall.     

Conserved V‐ATPase c subunit plays a role in plant growth by influencing V‐ATPase‐dependent endosomal trafficking

In plant cells, the vacuolar‐type H⁺‐ATPases (V‐ATPase) are localized in the tonoplast, Golgi, trans‐Golgi network and endosome. However, little is known about how V‐ATPase influences plant growth, particularly with regard to the V‐ATPase c subunit (VHA‐c). Here, we characterized the function of a VHA‐c gene from Puccinellia tenuiflora (PutVHA‐c) in plant growth. Compared to the wild‐type, transgenic plants overexpressing PutVHA‐c in Arabidopsis thaliana exhibit better growth phenotypes in root length, fresh weight, plant height and silique number under the normal and salt stress conditions due to noticeably higher V‐ATPase activity. Consistently, the Arabidopsis atvha‐c5 mutant shows reduced V‐ATPase activity and retarded plant growth. Furthermore, confocal and immunogold electron microscopy assays demonstrate that PutVHA‐c is mainly localized to endosomal compartments. The treatment of concanamycin A (ConcA), a specific inhibitor of V‐ATPases, leads to obvious aggregation of the endosomal compartments labelled with PutVHA‐c‐GFP. Moreover, ConcA treatment results in the abnormal localization of two plasma membrane (PM) marker proteins Pinformed 1 (AtPIN1) and regulator of G protein signalling‐1 (AtRGS1). These findings suggest that the decrease in V‐ATPase activity blocks endosomal trafficking. Taken together, our results strongly suggest that the PutVHA‐c plays an important role in plant growth by influencing V‐ATPase‐dependent endosomal trafficking.     

Nitrate induction of root hair density is mediated by TGA1/TGA4 and CPC transcription factors in Arabidopsis thaliana

Root hairs are specialized cells that are important for nutrient uptake. It is well established that nutrients such as phosphate have a great influence on root hair development in many plant species. Here we investigated the role of nitrate on root hair development at a physiological and molecular level. We showed that nitrate increases root hair density in Arabidopsis thaliana. We found that two different root hair defective mutants have significantly less nitrate than wild‐type plants, suggesting that in A. thaliana root hairs have an important role in the capacity to acquire nitrate. Nitrate reductase‐null mutants exhibited nitrate‐dependent root hair phenotypes comparable with wild‐type plants, indicating that nitrate is the signal that leads to increased formation of root hairs. We examined the role of two key regulators of root hair cell fate, CPC and WER, in response to nitrate treatments. Phenotypic analyses of these mutants showed that CPC is essential for nitrate‐induced responses of root hair development. Moreover, we showed that NRT1.1 and TGA1/TGA4 are required for pathways that induce root hair development by suppression of longitudinal elongation of trichoblast cells in response to nitrate treatments. Our results prompted a model where nitrate signaling via TGA1/TGA4 directly regulates the CPC root hair cell fate specification gene to increase formation of root hairs in A. thaliana.     

Phospholipase D and retromer promote recycling of TRPL ion channel via the endoplasmic reticulum

Plasma membrane protein trafficking is of fundamental importance for cell function and cell integrity of neurons and includes regulated protein recycling. In this work, we report a novel role of the endoplasmic reticulum (ER) for protein recycling as discovered in trafficking studies of the ion channel TRPL in photoreceptor cells of Drosophila. TRPL is located within the rhabdomeric membrane from where it is endocytosed upon light stimulation and stored in the cell body. Conventional immunohistochemistry as well as stimulated emission depletion super-resolution microscopy revealed TRPL storage at the ER after illumination, suggesting an unusual recycling route of TRPL. Our results also imply that both phospholipase D (PLD) and retromer complex are required for correct recycling of TRPL to the rhabdomeric membrane. Loss of PLD activity in PLD^3.1 mutants results in enhanced degradation of TRPL. In the retromer mutant vps35^MH20, TRPL is trapped in a Rab5-positive compartment. Evidenced by epistatic analysis in the double mutant PLD^3.1 vps35^MH20, PLD activity precedes retromer function. We propose a model in which PLD and retromer function play key roles in the transport of TRPL to an ER enriched compartment.     

Loss-of-Function Mutations of Retromer Large Subunit Genes Suppress the Phenotype of an Arabidopsis zig Mutant That Lacks Qb-SNARE VTI11

Arabidopsis thaliana zigzag (zig) is a loss-of-function mutant of Qb-SNARE VTI11, which is involved in membrane trafficking between the trans-Golgi network and the vacuole. zig-1 exhibits abnormalities in shoot gravitropism and morphology. Here, we report that loss-of-function mutants of the retromer large subunit partially suppress the zig-1 phenotype. Moreover, we demonstrate that three paralogous VPS35 genes of Arabidopsis have partially overlapping but distinct genetic functions with respect to zig-1 suppression. Tissue-specific complementation experiments using an endodermis-specific SCR promoter show that expression of VPS35B or VPS35C cannot complement the function of VPS35A. The data suggest the existence of functionally specialized paralogous VPS35 genes that nevertheless share common functions.     

Direct visualization of Agrobacterium‐delivered VirE2 in recipient cells

Agrobacterium tumefaciens is a natural genetic engineer widely used to deliver DNA into various recipients, including plant, yeast and fungal cells. The bacterium can transfer single‐stranded DNA molecules (T–DNAs) and bacterial virulence proteins, including VirE2. However, neither the DNA nor the protein molecules have ever been directly visualized after the delivery. In this report, we adopted a split‐GFP approach: the small GFP fragment (GFP11) was inserted into VirE2 at a permissive site to create the VirE2‐GFP11 fusion, which was expressed in A. tumefaciens; and the large fragment (GFP1–10) was expressed in recipient cells. Upon delivery of VirE2‐GFP11 into the recipient cells, GFP fluorescence signals were visualized. VirE2‐GFP11 was functional like VirE2; the GFP fusion movement could indicate the trafficking of Agrobacterium‐delivered VirE2. As the natural host, all plant cells seen under a microscope received the VirE2 protein in a leaf‐infiltration assay; most of VirE2 moved at a speed of 1.3–3.1 μm sec^?1 in a nearly linear direction, suggesting an active trafficking process. Inside plant cells, VirE2‐GFP formed filamentous structures of different lengths, even in the absence of T‐DNA. As a non‐natural host recipient, 51% of yeast cells received VirE2, which did not move inside yeast. All plant cells seen under a microscope transiently expressed the Agrobacterium‐delivered transgene, but only 0.2% yeast cells expressed the transgene. This indicates that Agrobacterium is a more efficient vector for protein delivery than T‐DNA transformation for a non‐natural host recipient: VirE2 trafficking is a limiting factor for the genetic transformation of a non‐natural host recipient. The split‐GFP approach could enable the real‐time visualization of VirE2 trafficking inside recipient cells.     

A root chicory MADS box sequence and the Arabidopsis flowering repressor FLC share common features that suggest conserved function in vernalization and de‐vernalization responses

Root chicory (Cichorium intybus var. sativum) is a biennial crop, but is harvested to obtain root inulin at the end of the first growing season before flowering. However, cold temperatures may vernalize seeds or plantlets, leading to incidental early flowering, and hence understanding the molecular basis of vernalization is important. A MADS box sequence was isolated by RT‐PCR and named FLC‐LIKE1 (CiFL1) because of its phylogenetic positioning within the same clade as the floral repressor Arabidopsis FLOWERING LOCUS C (AtFLC). Moreover, over‐expression of CiFL1 in Arabidopsis caused late flowering and prevented up‐regulation of the AtFLC target FLOWERING LOCUS T by photoperiod, suggesting functional conservation between root chicory and Arabidopsis. Like AtFLC in Arabidopsis, CiFL1 was repressed during vernalization of seeds or plantlets of chicory, but repression of CiFL1 was unstable when the post‐vernalization temperature was favorable to flowering and when it de‐vernalized the plants. This instability of CiFL1 repression may be linked to the bienniality of root chicory compared with the annual lifecycle of Arabidopsis. However, re‐activation of AtFLC was also observed in Arabidopsis when a high temperature treatment was used straight after seed vernalization, eliminating the promotive effect of cold on flowering. Cold‐induced down‐regulation of a MADS box floral repressor and its re‐activation by high temperature thus appear to be conserved features of the vernalization and de‐vernalization responses in distant species.     

Trafficking of Vacuolar Proteins: The Crucial Role of Arabidopsis Vacuolar Protein Sorting 29 in Recycling Vacuolar Sorting Receptor

The retromer is involved in recycling lysosomal sorting receptors in mammals. A component of the retromer complex in Arabidopsis thaliana, vacuolar protein sorting 29 (VPS29), plays a crucial role in trafficking storage proteins to protein storage vacuoles. However, it is not known whether or how vacuolar sorting receptors (VSRs) are recycled from the prevacuolar compartment (PVC) to the trans-Golgi network (TGN) during trafficking to the lytic vacuole (LV). Here, we report that VPS29 plays an essential role in the trafficking of soluble proteins to the LV from the TGN to the PVC. maigo1-1 (mag1-1) mutants, which harbor a knockdown mutation in VPS29, were defective in trafficking of two soluble proteins, Arabidopsis aleurain-like protein (AALP):green fluorescent protein (GFP) and sporamin:GFP, to the LV but not in trafficking membrane proteins to the LV or plasma membrane or via the secretory pathway. AALP:GFP and sporamin:GFP in mag1-1 protoplasts accumulated in the TGN but were also secreted into the medium. In mag1-1 mutants, VSR1 failed to recycle from the PVC to the TGN; rather, a significant proportion was transported to the LV; VSR1 overexpression rescued this defect. Moreover, endogenous VSRs were expressed at higher levels in mag1-1 plants. Based on these results, we propose that VPS29 plays a crucial role in recycling VSRs from the PVC to the TGN during the trafficking of soluble proteins to the LV.     

VPS29 is not an active metallo-phosphatase but is a rigid scaffold required for retromer interaction with accessory proteins

VPS29 is a key component of the cargo-binding core complex of retromer, a protein assembly with diverse roles in transport of receptors within the endosomal system. VPS29 has a fold related to metal-binding phosphatases and mediates interactions between retromer and other regulatory proteins. In this study we examine the functional interactions of mammalian VPS29, using X-ray crystallography and NMR spectroscopy. We find that although VPS29 can coordinate metal ions Mn(2+) and Zn(2+) in both the putative active site and at other locations, the affinity for metals is low, and lack of activity in phosphatase assays using a putative peptide substrate support the conclusion that VPS29 is not a functional metalloenzyme. There is evidence that structural elements of VPS29 critical for binding the retromer subunit VPS35 may undergo both metal-dependent and independent conformational changes regulating complex formation, however studies using ITC and NMR residual dipolar coupling (RDC) measurements show that this is not the case. Finally, NMR chemical shift mapping indicates that VPS29 is able to associate with SNX1 via a conserved hydrophobic surface, but with a low affinity that suggests additional interactions will be required to stabilise the complex in vivo. Our conclusion is that VPS29 is a metal ion-independent, rigid scaffolding domain, which is essential but not sufficient for incorporation of retromer into functional endosomal transport assemblies.     

Arabidopsis vacuolar sorting mutants (green fluorescent seed) can be identified efficiently by secretion of vacuole-targeted green fluorescent protein in their seeds

Two Arabidopsis thaliana genes have been shown to function in vacuolar sorting of seed storage proteins: a vacuolar sorting receptor, VSR1/ATELP1, and a retromer component, MAIGO1 (MAG1)/VPS29. Here, we show an efficient and simple method for isolating vacuolar sorting mutants of Arabidopsis. The method was based on two findings in this study. First, VSR1 functioned as a sorting receptor for beta-conglycinin by recognizing the vacuolar targeting signal. Second, when green fluorescent protein (GFP) fusion with the signal (GFP-CT24) was expressed in vsr1, mag1/vps29, and wild-type seeds, both vsr1and mag1/vps29 gave strongly fluorescent seeds but the wild type did not, suggesting that a defect in vacuolar sorting provided fluorescent seeds by the secretion of GFP-CT24 out of the cells. We mutagenized transformant seeds expressing GFP-CT24. From approximately 3,000,000 lines of M2 seeds, we obtained >100 fluorescent seeds and designated them green fluorescent seed (gfs) mutants. We report 10 gfs mutants, all of which caused missorting of storage proteins. We mapped gfs1 to VSR1, gfs2 to KAM2/GRV2, gfs10 to the At4g35870 gene encoding a novel membrane protein, and the others to different loci. This method should provide valuable insights into the complex molecular mechanisms underlying vacuolar sorting of storage proteins.     

Plant retromer, localized to the prevacuolar compartment and microvesicles in Arabidopsis, may interact with vacuolar sorting receptors

Receptors for acid hydrolases destined for the lytic compartment in yeast and mammalian cells are retrieved from intermediate, endosomal organelles with the help of a pentameric protein complex called the retromer. We cloned the Arabidopsis thaliana homologs of the three yeast proteins (Vps35, Vps29, and Vps26) constituting the larger subunit of retromer and prepared antisera against them. With these antibodies, we demonstrated the presence of a retromer-like protein complex in salt extracts prepared from Arabidopsis microsomes. This complex is associated with membranes that coequilibrate with prevacuolar compartment markers and with high-density sedimenting membranes. Immunogold negative staining identified these membranes as 90-nm-diameter coated microvesicles. Confocal laser scanning immunofluorescence studies performed on tobacco (Nicotiana tabacum) BY-2 cells revealed high degrees of colabeling between all three retromer antisera and the prevacuolar compartment (PVC) markers PEP12 and vacuolar sorting receptor VSR(At-1). The presence of plant retromer at the surface of multivesicular bodies was also demonstrated by immunogold labeling of sections obtained from high-pressure frozen/freeze-substituted specimens. Treatment of BY-2 cells with wortmannin led to swelling of the PVC and a separation of the VPS35 and VSR signals. Preliminary data suggesting that retromer interacts with the cytosolic domain of a VSR were obtained by immunoprecipitation experiments performed on detergent-solubilized microsomes with Vps35 antibodies.