Endosidin20 Targets the Cellulose Synthase Catalytic Domain to Inhibit Cellulose Biosynthesis.

Plant cellulose is synthesized by rosette-structured cellulose synthase (CESA) complexes (CSCs). Each CSC is composed of multiple subunits of CESAs representing three different isoforms. Individual CESA proteins contain conserved catalytic domains for catalyzing cellulose synthesis, other domains such as plant-conserved sequences, and class-specific regions that are thought to facilitate complex assembly and CSC trafficking. Because of the current lack of atomic-resolution structures for plant CSCs or CESAs, the molecular mechanism through which CESA catalyzes cellulose synthesis and whether its catalytic activity influences efficient CSC transport at the subcellular level remain unknown. Here, by performing chemical genetic analyses, biochemical assays, structural modeling, and molecular docking, we demonstrate that Endosidin20 (ES20) targets the catalytic site of CESA6 in Arabidopsis (Arabidopsis thaliana). Chemical genetic analysis revealed important amino acids that potentially participate in the catalytic activity of plant CESA6, in addition to previously identified conserved motifs across kingdoms. Using high spatiotemporal resolution live cell imaging, we found that inhibiting the catalytic activity of CESA6 by ES20 treatment reduced the efficiency of CSC transport to the plasma membrane. Our results demonstrate that ES20 is a chemical inhibitor of CESA activity and trafficking that represents a powerful tool for studying cellulose synthesis in plants.

Remove, Recycle, Degrade: Regulating Plasma Membrane Protein Accumulation.

Interactions between plant cells and the environment rely on modulation of protein receptors, transporters, channels, and lipids at the plasma membrane (PM) to facilitate intercellular communication, nutrient uptake, environmental sensing, and directional growth. These functions are fine-tuned by cellular pathways maintaining or reducing particular proteins at the PM. Proteins are endocytosed, and their fate is decided between recycling and degradation to modulate localization, abundance, and activity. Selective autophagy is another pathway regulating PM protein accumulation in response to specific conditions or developmental signals. The mechanisms regulating recycling, degradation, and autophagy have been studied extensively, yet we are just now addressing their regulation and coordination. Here, we (1) provide context concerning regulation of protein accumulation, recycling, or degradation by overviewing endomembrane trafficking; (2) discuss pathways regulating recycling and degradation in terms of cellular roles and cargoes; (3) review plant selective autophagy and its physiological significance; (4) focus on two decision-making mechanisms: regulation of recycling versus degradation of PM proteins and coordination between autophagy and vacuolar degradation; and (5) identify future challenges.

Interaction between VPS35 and RABG3f is necessary as a checkpoint to control fusion of late compartments with the vacuole.

Vacuoles are essential organelles in plants, playing crucial roles, such as cellular material degradation, ion and metabolite storage, and turgor maintenance. Vacuoles receive material via the endocytic, secretory, and autophagic pathways. Membrane fusion is the last step during which prevacuolar compartments (PVCs) and autophagosomes fuse with the vacuole membrane (tonoplast) to deliver cargoes. Protein components of the canonical intracellular fusion machinery that are conserved across organisms, including Arabidopsis thaliana, include complexes, such as soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs), that catalyze membrane fusion, and homotypic fusion and vacuole protein sorting (HOPS), that serve as adaptors which tether cargo vesicles to target membranes for fusion under the regulation of RAB-GTPases. The mechanisms regulating the recruitment and assembly of tethering complexes are not well-understood, especially the role of RABs in this dynamic regulation. Here, we report the identification of the small synthetic molecule Endosidin17 (ES17), which interferes with synthetic, endocytic, and autophagic traffic by impairing the fusion of late endosome compartments with the tonoplast. Multiple independent target identification techniques revealed that ES17 targets the VPS35 subunit of the retromer tethering complex, preventing its normal interaction with the Arabidopsis RAB7 homolog RABG3f. ES17 interference with VPS35-RABG3f interaction prevents the retromer complex to endosome anchoring, resulting in retention of RABG3f. Using multiple approaches, we show that VPS35-RABG3f-GTP interaction is necessary to trigger downstream events like HOPS complex assembly and fusion of late compartments with the tonoplast. Overall, our results support a role for the interaction of RABG3f-VPS35 as a checkpoint in the control of traffic toward the vacuole.

The Inhibitor Endosidin 4 Targets SEC7 Domain-Type ARF GTPase Exchange Factors and Interferes with Subcellular Trafficking in Eukaryotes.

The trafficking of subcellular cargos in eukaryotic cells crucially depends on vesicle budding, a process mediated by ARF-GEFs (ADP-ribosylation factor guanine nucleotide exchange factors). In plants, ARF-GEFs play essential roles in endocytosis, vacuolar trafficking, recycling, secretion, and polar trafficking. Moreover, they are important for plant development, mainly through controlling the polar subcellular localization of PIN-FORMED transporters of the plant hormone auxin. Here, using a chemical genetics screen in Arabidopsis thaliana, we identified Endosidin 4 (ES4), an inhibitor of eukaryotic ARF-GEFs. ES4 acts similarly to and synergistically with the established ARF-GEF inhibitor Brefeldin A and has broad effects on intracellular trafficking, including endocytosis, exocytosis, and vacuolar targeting. Additionally, Arabidopsis and yeast (Saccharomyces cerevisiae) mutants defective in ARF-GEF show altered sensitivity to ES4. ES4 interferes with the activation-based membrane association of the ARF1 GTPases, but not of their mutant variants that are activated independently of ARF-GEF activity. Biochemical approaches and docking simulations confirmed that ES4 specifically targets the SEC7 domain-containing ARF-GEFs. These observations collectively identify ES4 as a chemical tool enabling the study of ARF-GEF-mediated processes, including ARF-GEF-mediated plant development.

Chemical Screening Pipeline for Identification of Specific Plant Autophagy Modulators.

Autophagy is a major catabolic process in eukaryotes with a key role in homeostasis, programmed cell death, and aging. In plants, autophagy is also known to regulate agronomically important traits such as stress resistance, longevity, vegetative biomass, and seed yield. Despite its significance, there is still a shortage of reliable tools modulating plant autophagy. Here, we describe the first robust pipeline for identification of specific plant autophagy-modulating compounds. Our screening protocol comprises four phases: (1) high-throughput screening of chemical compounds in cell cultures of tobacco (Nicotiana tabacum); (2) confirmation of the identified hits in planta using Arabidopsis (Arabidopsis thaliana); (3) further characterization of the effect using conventional molecular biology methods; and (4) verification of chemical specificity on autophagy in planta. The methods detailed here streamline the identification of specific plant autophagy modulators and aid in unraveling the molecular mechanisms of plant autophagy.

Different Endomembrane Trafficking Pathways Establish Apical and Basal Polarities.

The endomembrane system is an interconnected network required to establish signal transduction, cell polarity, and cell shape in response to developmental or environmental stimuli. In the model plant Arabidopsis thaliana, there are numerous markers to visualize polarly localized plasma membrane proteins utilizing endomembrane trafficking. Previous studies have shown that the large ARF-GEF GNOM plays a key role in the establishment of basal (rootward) polarity, whereas the apically (shootward) polarized membrane proteins undergo sorting via different routes. However, the mechanism that maintains apical polarity is largely unknown. Here, we used a chemical genomic approach and identified the compound endosidin 16 (ES16), which perturbed apically localized plasma membrane proteins without affecting basal polarity. We demonstrated that ES16 is an inhibitor for recycling of apical, lateral, and nonpolar plasma membrane proteins as well as biosynthetic secretion, leaving the basal proteins as the only exceptions not subject to ES16 inhibition. Further evidence from pharmaceutical and genetic data revealed that ES16 effects are mediated through the regulation of small GTPase RabA proteins and that RabA GTPases work in concert with the BIG clade ARF-GEF to modulate the nonbasal trafficking. Our results reveal that ES16 defines a distinct pathway for endomembrane sorting routes and is essential for the establishment of cell polarity.

Endosidin2 targets conserved exocyst complex subunit EXO70 to inhibit exocytosis.

The exocyst complex regulates the last steps of exocytosis, which is essential to organisms across kingdoms. In humans, its dysfunction is correlated with several significant diseases, such as diabetes and cancer progression. Investigation of the dynamic regulation of the evolutionarily conserved exocyst-related processes using mutants in genetically tractable organisms such as Arabidopsis thaliana is limited by the lethality or the severity of phenotypes. We discovered that the small molecule Endosidin2 (ES2) binds to the EXO70 (exocyst component of 70 kDa) subunit of the exocyst complex, resulting in inhibition of exocytosis and endosomal recycling in both plant and human cells and enhancement of plant vacuolar trafficking. An EXO70 protein with a C-terminal truncation results in dominant ES2 resistance, uncovering possible distinct regulatory roles for the N terminus of the protein. This study not only provides a valuable tool in studying exocytosis regulation but also offers a potentially new target for drugs aimed at addressing human disease.

Comparative analysis reveals unexpected genome features of newly isolated Thraustochytrids strains: on ecological function and PUFAs biosynthesis.

BACKGROUND: Thraustochytrids are unicellular fungal-like marine protists with ubiquitous existence in marine environments. They are well-known for their ability to produce high-valued omega-3 polyunsaturated fatty acids (ω-3-PUFAs) (e.g., docosahexaenoic acid (DHA)) and hydrolytic enzymes. Thraustochytrid biomass has been estimated to surpass that of bacterioplankton in both coastal and oceanic waters indicating they have an important role in microbial food-web. Nevertheless, the molecular pathway and regulatory network for PUFAs production and the molecular mechanisms underlying ecological functions of thraustochytrids remain largely unknown. RESULTS: The genomes of two thraustochytrids strains (Mn4 and SW8) with ability to produce DHA were sequenced and assembled with a hybrid sequencing approach utilizing Illumina short paired-end reads and Pacific Biosciences long reads to generate a highly accurate genome assembly. Phylogenomic and comparative genomic analyses found that DHA-producing thraustochytrid strains were highly similar and possessed similar gene content. Analysis of the conventional fatty acid synthesis (FAS) and the polyketide synthase (PKS) systems for PUFAs production only detected incomplete and fragmentary pathways in the genome of these two strains. Surprisingly, secreted carbohydrate active enzymes (CAZymes) were found to be significantly depleted in the genomes of these 2 strains as compared to other sequenced relatives. Furthermore, these two strains possess an expanded gene repertoire for signal transduction and self-propelled movement, which could be important for their adaptations to dynamic marine environments. CONCLUSIONS: Our results demonstrate the possibility of a third PUFAs synthesis pathway besides previously described FAS and PKS pathways encoded in the genome of these two thraustochytrid strains. Moreover, lack of a complete set of hydrolytic enzymatic machinery for degrading plant-derived organic materials suggests that these two DHA-producing strains play an important role as a nutritional source rather than a nutrient-producer in marine microbial-food web. Results of this study suggest the existence of two types of saprobic thraustochytrids in the world's ocean. The first group, which does not produce cellulosic enzymes and live as 'left-over' scavenger of bacterioplankton, serves as a dietary source for the plankton of higher trophic levels and the other possesses capacity to live on detrital organic matters in the marine ecosystems.

Arabidopsis ribosomal proteins control vacuole trafficking and developmental programs through the regulation of lipid metabolism.

The vacuole is the most prominent compartment in plant cells and is important for ion and protein storage. In our effort to search for key regulators in the plant vacuole sorting pathway, ribosomal large subunit 4 (rpl4d) was identified as a translational mutant defective in both vacuole trafficking and normal development. Polysome profiling of the rpl4d mutant showed reduction in polysome-bound mRNA compared with wild-type, but no significant change in the general mRNA distribution pattern. Ribsomal profiling data indicated that genes in the lipid metabolism pathways were translationally down-regulated in the rpl4d mutant. Live imaging studies by Nile red staining suggested that both polar and nonpolar lipid accumulation was reduced in meristem tissues of rpl4d mutants. Pharmacological evidence showed that sterol and sphingolipid biosynthetic inhibitors can phenocopy the defects of the rpl4d mutant, including an altered vacuole trafficking pattern. Genetic evidence from lipid biosynthetic mutants indicates that alteration in the metabolism of either sterol or sphingolipid biosynthesis resulted in vacuole trafficking defects, similar to the rpl4d mutant. Tissue-specific complementation with key enzymes from lipid biosynthesis pathways can partially rescue both vacuole trafficking and auxin-related developmental defects in the rpl4d mutant. These results indicate that lipid metabolism modulates auxin-mediated tissue differentiation and endomembrane trafficking pathways downstream of ribosomal protein function.

An early secretory pathway mediated by GNOM-LIKE 1 and GNOM is essential for basal polarity establishment in Arabidopsis thaliana.

Spatial regulation of the plant hormone indole-3-acetic acid (IAA, or auxin) is essential for plant development. Auxin gradient establishment is mediated by polarly localized auxin transporters, including PIN-FORMED (PIN) proteins. Their localization and abundance at the plasma membrane are tightly regulated by endomembrane machinery, especially the endocytic and recycling pathways mediated by the ADP ribosylation factor guanine nucleotide exchange factor (ARF-GEF) GNOM. We assessed the role of the early secretory pathway in establishing PIN1 polarity in Arabidopsis thaliana by pharmacological and genetic approaches. We identified the compound endosidin 8 (ES8), which selectively interferes with PIN1 basal polarity without altering the polarity of apical proteins. ES8 alters the auxin distribution pattern in the root and induces a strong developmental phenotype, including reduced root length. The ARF-GEF-defective mutants gnom-like 1 (gnl1-1) and gnom (van7) are significantly resistant to ES8. The compound does not affect recycling or vacuolar trafficking of PIN1 but leads to its intracellular accumulation, resulting in loss of PIN1 basal polarity at the plasma membrane. Our data confirm a role for GNOM in endoplasmic reticulum (ER)-Golgi trafficking and reveal that a GNL1/GNOM-mediated early secretory pathway selectively regulates PIN1 basal polarity establishment in a manner essential for normal plant development.

The Conformation of a Plasma Membrane-Localized Somatic Embryogenesis Receptor Kinase Complex Is Altered by a Potato Aphid-Derived Effector.

Somatic embryogenesis receptor kinases (SERKs) are transmembrane receptors involved in plant immunity. Tomato (Solanum lycopersicum) carries three SERK members. One of these, SlSERK1, is required for Mi-1.2-mediated resistance to potato aphids (Macrosiphum euphorbiae). Mi-1.2 encodes a coiled-coil nucleotide-binding leucine-rich repeat protein that in addition to potato aphids confers resistance to two additional phloem-feeding insects and to root-knot nematodes (Meloidogyne spp.). How SlSERK1 participates in Mi-1.2-mediated resistance is unknown, and no Mi-1.2 cognate pest effectors have been identified. Here, we study the mechanistic involvement of SlSERK1 in Mi-1.2-mediated resistance. We show that potato aphid saliva and protein extracts induce the Mi-1.2 defense marker gene SlWRKY72b, indicating that both saliva and extracts contain a Mi-1.2 recognized effector. Resistant tomato cultivar Motelle (Mi-1.2/Mi-1.2) plants overexpressing SlSERK1 were found to display enhanced resistance to potato aphids. Confocal microscopy revealed that Mi-1.2 localizes at three distinct subcellular compartments: the plasma membrane, cytoplasm, and nucleus. Coimmunoprecipitation experiments in these tomato plants and in Nicotiana benthamiana transiently expressing Mi-1.2 and SlSERK1 showed that Mi-1.2 and SlSERK1 colocalize only in a microsomal complex. Interestingly, bimolecular fluorescence complementation analysis showed that the interaction of Mi-1.2 and SlSERK1 at the plasma membrane distinctively changes in the presence of potato aphid saliva, suggesting a model in which a constitutive complex at the plasma membrane participates in defense signaling upon effector binding.

Merging roads: chemical tools and cell biology to study unconventional protein secretion.

The endomembrane trafficking network is highly complex and dynamic, with both conventional and so-called unconventional routes which are in essence recently discovered pathways that are poorly understood in plants. One approach to dissecting endomembrane pathways that we have pioneered is the use of chemical biology. Classical genetic manipulations often deal with indirect pleiotropic phenotypes resulting from the perturbation of key players of the trafficking routes. Many of these difficulties can be circumvented using small molecules to modify or disrupt the function or localization of key proteins regulating these pathways. In this review, we summarize how small molecules have been used as probes to define these pathways, and how they could be used to increase current knowledge of unconventional protein secretion pathways.

Clusters of bioactive compounds target dynamic endomembrane networks in vivo.

Endomembrane trafficking relies on the coordination of a highly complex, dynamic network of intracellular vesicles. Understanding the network will require a dissection of cargo and vesicle dynamics at the cellular level in vivo. This is also a key to establishing a link between vesicular networks and their functional roles in development. We used a high-content intracellular screen to discover small molecules targeting endomembrane trafficking in vivo in a complex eukaryote, Arabidopsis thaliana. Tens of thousands of molecules were prescreened and a selected subset was interrogated against a panel of plasma membrane (PM) and other endomembrane compartment markers to identify molecules that altered vesicle trafficking. The extensive image dataset was transformed by a flexible algorithm into a marker-by-phenotype-by-treatment time matrix and revealed groups of molecules that induced similar subcellular fingerprints (clusters). This matrix provides a platform for a systems view of trafficking. Molecules from distinct clusters presented avenues and enabled an entry point to dissect recycling at the PM, vacuolar sorting, and cell-plate maturation. Bioactivity in human cells indicated the value of the approach to identifying small molecules that are active in diverse organisms for biology and drug discovery.

Nucleosome landscape and control of transcription in the human malaria parasite.

In eukaryotic cells, chromatin reorganizes within promoters of active genes to allow the transcription machinery and various transcription factors to access DNA. In this model, promoter-specific transcription factors bind DNA to initiate the production of mRNA in a tightly regulated manner. In the case of the human malaria parasite, Plasmodium falciparum, specific transcription factors are apparently underrepresented with regards to the size of the genome, and mechanisms underlying transcriptional regulation are controversial. Here, we investigate the modulation of DNA accessibility by chromatin remodeling during the parasite infection cycle. We have generated genome-wide maps of nucleosome occupancy across the parasite erythrocytic cycle using two complementary assays--the formaldehyde-assisted isolation of regulatory elements to extract protein-free DNA (FAIRE) and the MNase-mediated purification of mononucleosomes to extract histone-bound DNA (MAINE), both techniques being coupled to high-throughput sequencing. We show that chromatin architecture undergoes drastic upheavals throughout the parasite's cycle, contrasting with targeted chromatin reorganization usually observed in eukaryotes. Chromatin loosens after the invasion of the red blood cell and then repacks prior to the next cycle. Changes in nucleosome occupancy within promoter regions follow this genome-wide pattern, with a few exceptions such as the var genes involved in virulence and genes expressed at early stages of the cycle. We postulate that chromatin structure and nucleosome turnover control massive transcription during the erythrocytic cycle. Our results demonstrate that the processes driving gene expression in Plasmodium challenge the classical eukaryotic model of transcriptional regulation occurring mostly at the transcription initiation level.

A key role for vesicles in fungal secondary metabolism.

Eukaryotes have evolved highly conserved vesicle transport machinery to deliver proteins to the vacuole. In this study we show that the filamentous fungus Aspergillus parasiticus employs this delivery system to perform new cellular functions, the synthesis, compartmentalization, and export of aflatoxin; this secondary metabolite is one of the most potent naturally occurring carcinogens known. Here we show that a highly pure vesicle-vacuole fraction isolated from A. parasiticus under aflatoxin-inducing conditions converts sterigmatocystin, a late intermediate in aflatoxin synthesis, to aflatoxin B(1); these organelles also compartmentalize aflatoxin. The role of vesicles in aflatoxin biosynthesis and export was confirmed by blocking vesicle-vacuole fusion using 2 independent approaches. Disruption of A. parasiticus vb1 (encodes a protein homolog of AvaA, a small GTPase known to regulate vesicle fusion in A. nidulans) or treatment with Sortin3 (blocks Vps16 function, one protein in the class C tethering complex) increased aflatoxin synthesis and export but did not affect aflatoxin gene expression, demonstrating that vesicles and not vacuoles are primarily involved in toxin synthesis and export. We also observed that development of aflatoxigenic vesicles (aflatoxisomes) is strongly enhanced under aflatoxin-inducing growth conditions. Coordination of aflatoxisome development with aflatoxin gene expression is at least in part mediated by Velvet (VeA), a global regulator of Aspergillus secondary metabolism. We propose a unique 2-branch model to illustrate the proposed role for VeA in regulation of aflatoxisome development and aflatoxin gene expression.

MODIFIED VACUOLE PHENOTYPE1 is an Arabidopsis myrosinase-associated protein involved in endomembrane protein trafficking.

We identified an Arabidopsis (Arabidopsis thaliana) ethyl methanesulfonate mutant, modified vacuole phenotype1-1 (mvp1-1), in a fluorescent confocal microscopy screen for plants with mislocalization of a green fluorescent protein-delta tonoplast intrinsic protein fusion. The mvp1-1 mutant displayed static perinuclear aggregates of the reporter protein. mvp1 mutants also exhibited a number of vacuole-related phenotypes, as demonstrated by defects in growth, utilization of stored carbon, gravitropic response, salt sensitivity, and specific susceptibility to the fungal necrotroph Alternaria brassicicola. Similarly, crosses with other endomembrane marker fusions identified mislocalization to aggregate structures, indicating a general defect in protein trafficking. Map-based cloning showed that the mvp1-1 mutation altered a gene encoding a putative myrosinase-associated protein, and glutathione S-transferase pull-down assays demonstrated that MVP1 interacted specifically with the Arabidopsis myrosinase protein, THIOGLUCOSIDE GLUCOHYDROLASE2 (TGG2), but not TGG1. Moreover, the mvp1-1 mutant showed increased nitrile production during glucosinolate hydrolysis, suggesting that MVP1 may play a role in modulation of myrosinase activity. We propose that MVP1 is a myrosinase-associated protein that functions, in part, to correctly localize the myrosinase TGG2 and prevent inappropriate glucosinolate hydrolysis that could generate cytotoxic molecules.

Endosidin1 defines a compartment involved in endocytosis of the brassinosteroid receptor BRI1 and the auxin transporters PIN2 and AUX1.

Although it is known that proteins are delivered to and recycled from the plasma membrane (PM) via endosomes, the nature of the compartments and pathways responsible for cargo and vesicle sorting and cellular signaling is poorly understood. To define and dissect specific recycling pathways, chemical effectors of proteins involved in vesicle trafficking, especially through endosomes, would be invaluable. Thus, we identified chemicals affecting essential steps in PM/endosome trafficking, using the intensely localized PM transport at the tips of germinating pollen tubes. The basic mechanisms of this localized growth are likely similar to those of non-tip growing cells in seedlings. The compound endosidin 1 (ES1) interfered selectively with endocytosis in seedlings, providing a unique tool to dissect recycling pathways. ES1 treatment induced the rapid agglomeration of the auxin translocators PIN2 and AUX1 and the brassinosteroid receptor BRI1 into distinct endomembrane compartments termed "endosidin bodies"; however, the markers PIN1, PIN7, and other PM proteins were unaffected. Endosidin bodies were defined by the syntaxin SYP61 and the V-ATPase subunit VHA-a1, two trans-Golgi network (TGN)/endosomal proteins. Interestingly, brassinosteroid (BR)-induced gene expression was inhibited by ES1 and treated seedlings displayed a brassinolide (BL)-insensitive phenotype similar to a bri1 loss-of-function mutant. No effect was detected in auxin signaling. Thus, PIN2, AUX1, and BRI1 use interactive pathways involving an early SYP61/VHA-a1 endosomal compartment.

Label-Free Target Identification and Confirmation Using Thermal Stability Shift Assays.

Target identification presents one of the biggest challenges to chemical genomic approaches. In recent years, several methods have been applied for target identification and validation in plant cells. Here, we describe a label-free method based on the thermodynamic stabilization of a protein by interaction with a small-molecule ligand. With increasing temperature, proteins undergo thermal denaturation resulting in irreversible aggregation and precipitation. The binding of a small molecule to its target can enhance protein stability resulting in an increased temperature of aggregation (Tagg). This distinct increase in the temperature of aggregation known as a thermal shift can identify a compound-target protein interaction in high-throughput assays or, validate a predicted interaction.

The use of chemical genomics to investigate pathways intersecting auxin-dependent responses and endomembrane trafficking in Arabidopsis thaliana.

Plant endomembrane system is essential for viability and necessary for proper development and signal transduction signal processes. Links between the endomembrane system and auxin signaling have been reported by classical genetics screens. However, the relationship between these processes is not well understood. Chemical genomics is a powerful approach to dissect various processes overcoming lethality and redundancy issues. This approach uses small molecules to modify or disrupt the function of specific proteins and biological processes. We present a screen in Arabidopsis thaliana to identify compound affecting auxin-dependent responses and components of the endomembrane system. A gravitropic-response based screen is performed in Arabidopsis seedlings. The identified gravitropic effectors are tested in terms of auxin responsiveness and their effects on endomembrane compartments. These bioactive compounds will be valuable tools for dissecting endomembrane trafficking and auxin signaling processes.