Transport Of Ricin Research Articles

Modulation of Ricin Intoxication by the Autophagy Inhibitor EACC.

The compound EACC (ethyl (2-(5-nitrothiophene-2-carboxamido) thiophene-3-carbonyl) carbamate) was recently reported to inhibit fusion of autophagosomes with lysosomes in a reversible manner by inhibiting recruitment of syntaxin 17 to autophagosomes. We report here that this compound also provides a strong protection against the protein toxin ricin as well as against other plant toxins such as abrin and modeccin. The protection did not seem to be caused by inhibition of endocytosis and retrograde transport, but rather by inhibited release of the enzymatically active A-moiety to the cytosol. The TANK-binding kinase 1 (TBK1) has been reported to phosphorylate syntaxin 17 and be required for initiation of autophagy. The inhibitor of TBK1, MRT68601, induced in itself a strong sensitization to ricin, apparently by increasing transport to the Golgi apparatus. Importantly, MRT68601 increased Golgi transport of ricin even in the presence of EACC, but EACC was still able to inhibit intoxication, supporting the idea that EACC protects at a late step along the retrograde pathway. These results also indicate that phosphorylation of syntaxin 17 is not required for the protection observed.

Structural Analysis of Toxin-Neutralizing, Single-Domain Antibodies that Bridge Ricin’s A-B Subunit Interface

Ricin toxin kills mammalian cells with notorious efficiency. The toxin’s B subunit (RTB) is a Gal/GalNAc-specific lectin that attaches to cell surfaces and promotes retrograde transport of ricin’s A subunit (RTA) to the trans Golgi network (TGN) and endoplasmic reticulum (ER). RTA is liberated from RTB in the ER and translocated into the cell cytoplasm, where it functions as a ribosome-inactivating protein. While antibodies against ricin’s individual subunits have been reported, we now describe seven alpaca-derived, single-domain antibodies (VHHs) that span the RTA-RTB interface, including four Tier 1 VHHs with IC50 values <1 nM. Crystal structures of each VHH bound to native ricin holotoxin revealed three different binding modes, based on contact with RTA’s F-G loop (mode 1), RTB’s subdomain 2γ (mode 2) or both (mode 3). VHHs in modes 2 and 3 were highly effective at blocking ricin attachment to HeLa cells and immobilized asialofetuin, due to framework residues (FR3) that occupied the 2γ Gal/GalNAc-binding pocket and mimic ligand. The four Tier 1 VHHs also interfered with intracellular functions of RTB, as they neutralized ricin in a post-attachment cytotoxicity assay (e.g., the toxin was bound to cell surfaces before antibody addition) and reduced the efficiency of toxin transport to the TGN. We conclude that the RTA-RTB interface is a target of potent toxin-neutralizing antibodies that interfere with both extracellular and intracellular events in ricin’s cytotoxic pathway.

Diacylglycerol kinase and phospholipase D inhibitors alter the cellular lipidome and endosomal sorting towards the Golgi apparatus

The membrane lipids diacylglycerol (DAG) and phosphatidic acid (PA) are important second messengers that can regulate membrane transport by recruiting proteins to the membrane and by altering biophysical membrane properties. DAG and PA are involved in the transport from the Golgi apparatus to endosomes, and we have here investigated whether changes in these lipids might be important for regulation of transport to the Golgi using the protein toxin ricin. Modulation of DAG and PA levels using DAG kinase (DGK) and phospholipase D (PLD) inhibitors gave a strong increase in retrograde ricin transport, but had little impact on ricin recycling or degradation. Inhibitor treatment strongly affected the endosome morphology, increasing endosomal tubulation and size. Furthermore, ricin was present in these tubular structures together with proteins known to regulate retrograde transport. Using siRNA to knock down different isoforms of PLD and DGK, we found that several isoforms of PLD and DGK are involved in regulating ricin transport to the Golgi. Finally, by performing lipidomic analysis we found that the DGK inhibitor gave a weak, but expected, increase in DAG levels, while the PLD inhibitor gave a strong and unexpected increase in DAG levels, showing that it is important to perform lipidomic analysis when using inhibitors of lipid metabolism.

The anti-tumor drug 2-hydroxyoleic acid (Minerval) stimulates signaling and retrograde transport.

2-hydroxyoleic acid (OHOA, Minerval®) is an example of a substance used for membrane lipid therapy, where the cellular membranes rather than specific proteins constitute the therapeutical target. OHOA is thought to mediate its anti-tumor effect by affecting the biophysical properties of membranes, which leads to altered recruitment and activation of amphitropic proteins, altered cellular signaling, and eventual cell death. Little is known about the initial signaling events upon treatment with OHOA, and whether the altered membrane properties would have any impact on the dynamic intracellular transport system. In the present study we demonstrate that treatment with OHOA led to a rapid release of intracellular calcium and activation of multiple signaling pathways in HeLa cells, including the PI3K-AKT1-MTOR pathway and several MAP kinases, in a process independent of the EGFR. By lipidomics we confirmed that OHOA was incorporated into several lipid classes. Concomitantly, OHOA potently increased retrograde transport of the plant toxin ricin from endosomes to the Golgi and further to the endoplasmic reticulum. The OHOA-stimulated ricin transport seemed to require several amphitropic proteins, including Src, phospholipase C, protein kinase C, and also Ca2+/calmodulin. Interestingly, OHOA induced a slight increase in endosomal localization of the retromer component VPS35. Thus, our data show that addition of a lipid known to alter membrane properties not only affects signaling, but also intracellular transport.

A Bispecific Antibody Promotes Aggregation of Ricin Toxin on Cell Surfaces and Alters Dynamics of Toxin Internalization and Trafficking.

JJX12 is an engineered bispecific antibody against ricin, a member of the medically important A-B family of toxins that exploits retrograde transport as means to gain entry into the cytosol of target cells. JJX12 consists of RTA-D10, a camelid single variable domain (VHH) antibody directed against an epitope on ricin’s enzymatic subunit (RTA), linked via a 15-mer peptide to RTB-B7, a VHH against ricin’s bivalent galactose binding subunit (RTB). We previously reported that JJX12, but not an equimolar mixture of RTA-D10 and RTB-B7 monomers, was able to passively protect mice against a lethal dose ricin challenge, demonstrating that physically linking RTB-B7 and RTA-D10 is critical for toxin-neutralizing activity in vivo. We also reported that JJX12 promotes aggregation of ricin in solution, presumably through the formation of intermolecular crosslinking. In the current study, we now present evidence that JJX12 affects the dynamics of ricin uptake and trafficking in human epithelial cells. Confocal microscopy, as well as live cell imaging coupled with endocytosis pathway-specific inhibitors, revealed that JJX12-toxin complexes are formed on the surfaces of mammalian cells and internalized via a pathway sensitive to amiloride, a known inhibitor of macropinocytosis. Moreover, in the presence of JJX12, retrograde transport of ricin to the trans-Golgi network was significantly reduced, while accumulation of the toxin in late endosomes was significantly enhanced. In summary, we propose that JJX12, by virtue of its ability to crosslink ricin toxin, alters the route of toxin uptake and trafficking within cells.

The role of EDEM2 compared with EDEM1 in ricin transport from the endoplasmic reticulum to the cytosol

EDEM1 [ER (endoplasmic reticulum)-degradation-enhancing α-mannosidase I-like protein 1] and EDEM2 are crucial regulators of ERAD (ER-associated degradation) that extracts non-native glycoproteins from the calnexin chaperone system. Ricin is a potent plant cytotoxin composed of an A-chain (RTA) connected by a disulfide bond to a cell-binding lectin B-chain (RTB). After endocytic uptake, the toxin is transported retrogradely to the ER, where the enzymatically active RTA is translocated to the cytosol in a similar manner as misfolded ER proteins. This transport is promoted by EDEM1. In the present study we report that EDEM2 is also involved in ricin retrotranslocation out of the ER. However, the role of EDEM1 and EDEM2in ricin transport to the cytosol seems to differ. EDEM2 promotes ricin retrotranslocation irrespectively of ER translocon accessibility; moreover, co-immunoprecipitation and pull-down studies revealed that more ricin can interact with EDEM2 in comparison with EDEM1. On the other hand, interactions of both lectins with RTA are dependent on the structure of the RTA. Thus our data display a newly discovered role for EDEM2. Moreover, analysis of the involvement of EDEM1 and EDEM2 in ricin retrotranslocation to the cytosol may provide crucial information about general mechanisms of the recognition of ERAD substrates in the ER.

BiP Negatively Affects Ricin Transport

The AB plant toxin ricin binds both glycoproteins and glycolipids at the cell surface via its B subunit. After binding, ricin is endocytosed and then transported retrogradely through the Golgi to the endoplasmic reticulum (ER). In the ER, the A subunit is retrotranslocated to the cytosol in a chaperone-dependent process, which is not fully explored. Recently two separate siRNA screens have demonstrated that ER chaperones have implications for ricin toxicity. ER associated degradation (ERAD) involves translocation of misfolded proteins from ER to cytosol and it is conceivable that protein toxins exploit this pathway. The ER chaperone BiP is an important ER regulator and has been implicated in toxicity mediated by cholera and Shiga toxin. In this study, we have investigated the role of BiP in ricin translocation to the cytosol. We first show that overexpression of BiP inhibited ricin translocation and protected cells against the toxin. Furthermore, shRNA-mediated depletion of BiP enhanced toxin translocation resulting in increased cytotoxicity. BiP-dependent inhibition of ricin toxicity was independent of ER stress. Our findings suggest that in contrast to what was shown with the Shiga toxin, the presence of BiP does not facilitate, but rather inhibits the entry of ricin into the cytosol.

Antibody to Ricin A Chain Hinders Intracellular Routing of Toxin and Protects Cells Even after Toxin Has Been Internalized

BackgroundMechanisms of antibody-mediated neutralization are of much interest. For plant and bacterial A-B toxins, A chain mediates toxicity and B chain binds target cells. It is generally accepted and taught that antibody (Ab) neutralizes by preventing toxin binding to cells. Yet for some toxins, ricin included, anti-A chain Abs afford greater protection than anti-B. The mechanism(s) whereby Abs to the A chain neutralize toxins are not understood.Methodology/Principal FindingsWe use quantitative confocal imaging, neutralization assays, and other techniques to study how anti-A chain Abs function to protect cells. Without Ab, ricin enters cells and penetrates to the endoplasmic reticulum within 15 min. Within 45–60 min, ricin entering and being expelled from cells reaches equilibrium. These results are consistent with previous observations, and support the validity of our novel methodology. The addition of neutralizing Ab causes ricin accumulation at the cell surface, delays internalization, and postpones retrograde transport of ricin. Ab binds ricin for >6hr as they traffic together through the cell. Ab protects cells even when administered hours after exposure.Conclusions/Key FindingsWe demonstrate the dynamic nature of the interaction between the host cell and toxin, and how Ab can alter the balance in favor of the cell. Ab blocks ricin’s entry into cells, hinders its intracellular routing, and can protect even after ricin is present in the target organelle, providing evidence that the major site of neutralization is intracellular. These data add toxins to the list of pathogenic agents that can be neutralized intracellularly and explain the in vivo efficacy of delayed administration of anti-toxin Abs. The results encourage the use of post-exposure passive Ab therapy, and show the importance of the A chain as a target of Abs.

Ricin and Ricin-Containing Immunotoxins: Insights into Intracellular Transport and Mechanism of action in Vitro

Ricin is a type II ribosome inactivating protein (RIP) isolated from castor beans. Its high toxicity classifies it as a possible biological weapon. On the other hand, ricin linked to specific monoclonal antibodies or used in other conjugates has powerful medical applications. Ricin consists of an A-chain (RTA) that damages ribosomes and inhibits protein synthesis, and a B-chain that plays a role in binding and cellular uptake. A number of recent studies have demonstrated that ricin-induced inhibition of protein synthesis is not the only mechanism responsible for cell death. It turns out that ricin is able to induce apoptosis in different cell lines and multiple organs in animals. However, the molecular link between protein synthesis inhibition and ricin-dependent triggering of apoptotic cell death is unclear. This review describes the intracellular transport of ricin and ricin-based immunotoxins and their mechanism of action in different non-malignant and cancer cell lines. Moreover, various ricin-containing immunotoxins, their composition, medical applications and side-effects will be described and discussed. Understanding the mechanism of action of ricin-based immunotoxins will facilitate construction of effectively acting immunotoxins that can be used in the clinic for cancer treatment.

Intracellular Transport of Plant Toxins Ricin and Viscumin from Different Plasma Membrane Sites

Binding of ricin and viscumin to paraformaldehyde fixed cell surface has been studied by confocal laser scanning microscopy (CLSM). Both toxins were labeled with different fluorochromes to allow for their identification after being applied jointly. The experiments indicated that viscumin and ricin bind to different cell receptors. Viscumin bound to the very periphery of the cells including lamellapodia and cell contact regions. Labeled ricin became localized in surface clusters located close to the cell body. The binding of toxins to the cell membrane was completely inhibited by 100 mmol/l lactose and in the presence of unlabeled homological toxins 500 times in abundance of the fluorochromed toxins. The experiments indicate that uptake and intracellular transport of ricin and viscumin starts from different membrane sites.

Role of Phospholipase A2 in Retrograde Transport of Ricin

Ricin is a protein toxin classified as a bioterror agent, for which there are no known treatment options available after intoxication. It is composed of an enzymatically active A-chain connected by a disulfide bond to a cell binding B-chain. After internalization by endocytosis, ricin is transported retrogradely to the Golgi and ER, from where the ricin A-chain is translocated to the cytosol where it inhibits protein synthesis and thus induces cell death. We have identified cytoplasmic phospholipase A2 (PLA2) as an important factor in ricin retrograde transport. Inhibition of PLA2 protects against ricin challenge, however the toxin can still be endocytosed and transported to the Golgi. Interestingly, ricin transport from the Golgi to the ER is strongly impaired in response to PLA2 inhibition. Confocal microscopy analysis shows that ricin is still colocalized with the trans-Golgi marker TGN46 in the presence of PLA2 inhibitor, but less is colocalized with the cis-Golgi marker GM130. We propose that PLA2 inhibition results in impaired ricin transport through the Golgi stack, thus preventing it from reaching the ER. Consequently, ricin cannot be translocated to the cytosol to exert its toxic action.

Development of a quantitative RT-PCR assay to examine the kinetics of ribosome depurination by ribosome inactivating proteins using Saccharomyces cerevisiae as a model

Ricin produced by the castor bean plant and Shiga toxins produced by pathogenic Escherichia coli (STEC) and Shigella dysenteriae are type II ribosome inactivating proteins (RIPs), containing an enzymatically active A subunit that inhibits protein synthesis by removing an adenine from the α-sarcin/ricin loop (SRL) of the 28S rRNA. There are currently no known antidotes to Shiga toxin or ricin, and the ability to screen large chemical libraries for inhibitors has been hindered by lack of quantitative assays for catalytic activity that can be adapted to a high throughput format. Here, we describe the development of a robust and quantitative reverse transcription polymerase chain reaction (qRT-PCR) assay that can directly measure the toxins' catalytic activity on ribosomes and can be used to examine the kinetics of depurination in vivo. The qRT-PCR assay exhibited a much wider dynamic range than the previously used primer extension assay (500-fold vs. 16-fold) and increased sensitivity (60 pM vs. 0.57 nM). Using this assay, a 400-fold increase in ribosome depurination was observed in yeast expressing ricin A chain (RTA) relative to uninduced cells. Pteroic acid, a known inhibitor of enzymatic activity, inhibited ribosome depurination by RTA and Shiga toxin 2 with an IC(50) of ∼ 100 μM, while inhibitors of ricin transport failed to inhibit catalytic activity. These results demonstrate that the qRT-PCR assay would enable refined kinetic studies with RIPs and could be a powerful screening tool to identify inhibitors of catalytic activity.

Interplay between Toxin Transport and Flotillin Localization

The flotillin proteins are localized in lipid domains at the plasma membrane as well as in intracellular compartments. In the present study, we examined the importance of flotillin-1 and flotillin-2 for the uptake and transport of the bacterial Shiga toxin (Stx) and the plant toxin ricin and we investigated whether toxin binding and uptake were associated with flotillin relocalization. We observed a toxin-induced redistribution of the flotillins, which seemed to be regulated in a p38-dependent manner. Our experiments provide no evidence for a changed endocytic uptake of Stx or ricin in cells silenced for flotillin-1 or -2. However, the Golgi-dependent sulfation of both toxins was significantly reduced in flotillin knockdown cells. Interestingly, when the transport of ricin to the ER was investigated, we obtained an increased mannosylation of ricin in flotillin-1 and flotillin-2 knockdown cells. The toxicity of both toxins was twofold increased in flotillin-depleted cells. Since BFA (Brefeldin A) inhibits the toxicity even in flotillin knockdown cells, the retrograde toxin transport is apparently still Golgi-dependent. Thus, flotillin proteins regulate and facilitate the retrograde transport of Stx and ricin.

Sorting nexin 8 regulates endosome-to-Golgi transport

Sorting nexin 8 (SNX8) belongs to the sorting nexin protein family, whose members are involved in endocytosis and endosomal sorting and signaling. The function of SNX8 has so far been unknown. Here, we have investigated the role of SNX8 in intracellular transport of the bacterial toxin Shiga toxin (Stx) and the plant toxin ricin. After being endocytosed, these toxins are transported retrogradely from endosomes, via the Golgi apparatus and the endoplasmic reticulum (ER), into the cytosol, where they exert their toxic effect. Interestingly, our experiments show that SNX8 regulates the transport of Stx and ricin differently; siRNA-mediated knockdown of SNX8 significantly increased the Stx transport to the trans-Golgi network (TGN), whereas ricin transport was slightly inhibited. We also found that SNX8 colocalizes with early endosome antigen 1 (EEA1) and with retromer components, suggesting an endosomal localization of SNX8 and supporting our finding that SNX8 is involved in endosomal sorting.

Cellular Trafficking of Quantum Dot-Ligand Bioconjugates and Their Induction of Changes in Normal Routing of Unconjugated Ligands

Can quantum dots (Qdots) act as relevant intracellular probes to investigate routing of ligands in live cells? The intracellular trafficking of Qdots that were coupled to the plant toxin ricin, Shiga toxin, or the ligand transferrin (Tf) was studied by confocal fluorescence microscopy. The Tf:Qdots were internalized by clathrin-dependent endocytosis as fast as Tf, but their recycling was blocked. Unlike Shiga toxin, the Shiga:Qdot bioconjugate was not routed to the Golgi apparatus. The internalized ricin:Qdot bioconjugates localized to the same endosomes as ricin itself but could not be visualized in the Golgi apparatus. Importantly, we find that the endosomal accumulation of ricin:Qdots affects endosome-to-Golgi transport of both ricin and Shiga toxin: Transport of ricin was reduced whereas transport of Shiga toxin was increased. In conclusion, the data reveal that, although coupling of Qdots to a ligand does not necessarily change the endocytic pathway normally used by the ligands studied, it appears that the ligand-coupled Qdot nanoparticles can be arrested within endosomes and somehow perturb the normal endosomal sorting in cells. Thus, the results demonstrate that Qdots may have severe consequences on cell physiology.

Phosphoinositide‐Regulated Retrograde Transport of Ricin: Crosstalk Between hVps34 and Sorting Nexins

The plant toxin ricin is transported from the plasma membrane via early endosomes and the Golgi apparatus to the endoplasmic reticulum. From this compartment, it enters the cytosol and inhibits protein synthesis. Lipid phosphorylation is an important regulator of vesicular transport, and in the present study we have investigated the role of the phosphatidylinositol (PI) 3-kinase hVps34 in retrograde transport of ricin. Our data demonstrate that transport of ricin from endosomes to the Golgi apparatus in human embryonic kidney cells (HEK 293) is dependent on PI(3)P. By using PI 3-kinase inhibitors, by sequestering the hVps34 product PI(3)P and by expressing mutants of hVps34 or small interfering RNA targeted against its messenger RNA, we show that hVps34 and its product PI(3)P are involved in transport of ricin from endosome to Golgi apparatus. Furthermore, we identify two effector proteins in the hVps34-dependent pathway, namely sorting nexin (SNX) 2 and SNX4. Knockdown of SNX2 or SNX4 inhibits ricin transport to the Golgi apparatus to the same extent as when hVps34 is perturbed. Furthermore, inhibition or knockdown of hVps34 redistributes these proteins. Interestingly, knocking down both SNX2 and SNX4 results in a better inhibition than knocking down only one of them, suggesting that they may act on separate pathways.

Depletion of Sphingolipids Facilitates Endosome to Golgi Transport of Ricin

It has been previously demonstrated that depletion of cholesterol inhibits endosome to Golgi transport. Whether this inhibition is due to disruption of sphingolipid- and cholesterol-containing lipid rafts that are selected for Golgi transport or whether there is a physical requirement of cholesterol for either membrane deformations, facilitating formation of transport vesicles, or for recruitment of cytosolic constituents is not obvious. To investigate this in more detail, we have studied endosome to Golgi transport of ricin in sphingolipid-deficient cells using either a mutant cell line that does not express serine palmitoyltransferase, the first enzyme in sphingolipid biosynthesis, or a specific inhibitor, myriocin, of the same enzyme. Depletion of sphingolipids gave an increased sensitivity to ricin, and this increased sensitivity was inhibited by addition of sphingolipids. Importantly, endosome to Golgi transport of ricin, measured as sulfation of a modified ricin molecule, was increased in sphingolipid-deficient cells. No effect was seen on other pathways taken by ricin. Interestingly, cholesterol depletion inhibited endosome to Golgi transport even in cells with reduced levels of sphingolipids, suggesting that cholesterol as such is required for formation of transport vesicles. Our results indicate that the presence of sphingolipids actually limits and may function to control endosome to Golgi transport of ricin.

Transport of Ricin from Endosomes to the Golgi Apparatus is Regulated by Rab6A and Rab6A′

Ricin is transported from early endosomes and/or the recycling compartment to the trans-Golgi network (TGN) and subsequently to the endoplasmic recticulum (ER) before it enters the cytosol and intoxicates cells. We have investigated the role of the Rab6 isoforms in retrograde transport of ricin using both oligo- and vector-based RNAi assays. Ricin transport to the TGN was inhibited by the depletion of Rab6A when the Rab6A messenger RNA (mRNA) levels were reduced by more than 40% and less than 75%. However, when Rab6A mRNA was reduced by more than 75% and Rab6A' mRNA was simultaneously up-regulated, the inhibition of ricin sulfation was abolished, indicating that the up-regulation of Rab6A' may compensate for the loss of Rab6A function. In addition, we found that a near complete depletion of Rab6A' gave approximately 40% reduction in ricin sulfation. The up-regulation of Rab6A mRNA levels did not seem to compensate for the loss of Rab6A' function. The depletion of both Rab6A and Rab6A' gave a stronger inhibition of ricin sulfation than what was observed knocking down the two isoforms separately. In conclusion, both Rab6A and Rab6A' seem to be involved in the transport of ricin from endosomes to the Golgi apparatus.

Abducens internuclear neurons depend on their target motoneurons for survival during early postnatal development

The highly specific projection of abducens internuclear neurons onto medial rectus motoneurons in the oculomotor nucleus is a good model to evaluate the dependence on target cells for survival during development and in the adult. Thus, the procedure we chose to selectively deprive abducens internuclear neurons of their natural target was the enucleation of postnatal day 1 rats to induce the death of medial rectus motoneurons. Two months later, we evaluated both the extent of reduction in target size, by immunocytochemistry against choline acetyltransferase (ChAT) and Nissl counting, and the percentage of abducens internuclear neurons surviving target loss, by calretinin immunostaining and horseradish peroxidase (HRP) retrograde tracing. Firstly, axotomized oculomotor motoneurons died in a high percentage (∼80%) as visualized 2 months after lesion. In addition, we showed a transient (1 month) and reversible down-regulation of ChAT expression in extraocular motoneurons induced by injury. Secondly, 2 months after enucleation, 61.6% and 60.5% of the population of abducens internuclear neurons appeared stained by retrograde tracing and calretinin immunoreaction, respectively, indicating a significant extent of cell death after target loss (38.4% or 39.5%). By contrast, in the adult rat, neither extraocular motoneurons died in response to axotomy nor abducens internuclear neurons died due to the loss of their target motoneurons induced by the retrograde transport of toxic ricin injected in the medial rectus muscle. These results indicate that, during development, abducens internuclear neurons depend on their target motoneurons for survival, and that they lose this dependence with maturation.

Transport of ricin and 2S albumin precursors to the storage vacuoles of Ricinus communis endosperm involves the Golgi and VSR-like receptors.

We have studied the transport of proricin and pro2S albumin to the protein storage vacuoles of developing castor bean (Ricinus communis L.) endosperm. Immunoelectron microscopy and cell fractionation reveal that both proteins travel through the Golgi apparatus and co-localize throughout their route to the storage vacuole. En route to the PSV, the proteins co-localize in large (>200 nm) vesicles, which are likely to represent developing storage vacuoles. We further show that the sequence-specific vacuolar sorting signals of both proricin and pro2SA bind in vitro to proteins that have high sequence similarity to members of the VSR/AtELP/BP-80 vacuolar sorting receptor family, generally associated with clathrin-mediated traffic to the lytic vacuole. The implications of these findings in relation to the current model for protein sorting to storage vacuoles are discussed.