A classical swine fever virus E2 fusion protein produced in plants elicits a neutralizing humoral immune response in mice and pigs.

Classical swine fever (CSF) is one of the most important viral diseases of swine worldwide. Although live or attenuated virus vaccines have been used to control CSFV, it is difficult to distinguish vaccinated pigs from infected pigs; this leads to restrictions on import and export. Subunit vaccines based on the CSFV E2 glycoprotein have been developed using baculovirus or insect cell systems, but some weaknesses remain. Here, we describe production of an E2 recombinant protein using a Nicotiana benthamiana plant expression system. To do this, we took advantage of the ability of the swine Fc domain to increase solubility and stability of the fusion protein and to strengthen immune responses in target animals. N. benthamiana expressed high amounts of pFc2-fused E2 proteins, which were isolated and purified by affinity chromatography to yield a high pure recombinant protein in a cost-effective manner. Native-polyacrylamide gel electrophoresis and size exclusion chromatography confirmed that the pmE2:pFc2 fusion exists as a multimer rather than as a dimer. Injection of recombinant pmE2 protein into mice or piglets generated anti-pmE2 antibodies with efficient neutralizing activity against CSFV. These results suggest that a purified recombinant E2 protein produced in N. benthamiana generates high titers of neutralizing antibodies in vivo; as such, the protein could be developed as a subunit vaccine against CSFV.

Adaptor protein complex 2-mediated endocytosis is crucial for male reproductive organ development in Arabidopsis.

Fertilization in flowering plants requires the temporal and spatial coordination of many developmental processes, including pollen production, anther dehiscence, ovule production, and pollen tube elongation. However, it remains elusive as to how this coordination occurs during reproduction. Here, we present evidence that endocytosis, involving heterotetrameric adaptor protein complex 2 (AP-2), plays a crucial role in fertilization. An Arabidopsis thaliana mutant ap2m displays multiple defects in pollen production and viability, as well as elongation of staminal filaments and pollen tubes, all of which are pivotal processes needed for fertilization. Of these abnormalities, the defects in elongation of staminal filaments and pollen tubes were partially rescued by exogenous auxin. Moreover, DR5rev:GFP (for green fluorescent protein) expression was greatly reduced in filaments and anthers in ap2m mutant plants. At the cellular level, ap2m mutants displayed defects in both endocytosis of N-(3-triethylammonium-propyl)-4-(4-diethylaminophenylhexatrienyl) pyridinium dibromide, a lypophilic dye used as an endocytosis marker, and polar localization of auxin-efflux carrier PIN FORMED2 (PIN2) in the stamen filaments. Moreover, these defects were phenocopied by treatment with Tyrphostin A23, an inhibitor of endocytosis. Based on these results, we propose that AP-2-dependent endocytosis plays a crucial role in coordinating the multiple developmental aspects of male reproductive organs by modulating cellular auxin level through the regulation of the amount and polarity of PINs.

The immediate upstream region of the 5'-UTR from the AUG start codon has a pronounced effect on the translational efficiency in Arabidopsis thaliana.

The nucleotide sequence around the translational initiation site is an important cis-acting element for post-transcriptional regulation. However, it has not been fully understood how the sequence context at the 5'-untranslated region (5'-UTR) affects the translational efficiency of individual mRNAs. In this study, we provide evidence that the 5'-UTRs of Arabidopsis genes showing a great difference in the nucleotide sequence vary greatly in translational efficiency with more than a 200-fold difference. Of the four types of nucleotides, the A residue was the most favourable nucleotide from positions -1 to -21 of the 5'-UTRs in Arabidopsis genes. In particular, the A residue in the 5'-UTR from positions -1 to -5 was required for a high-level translational efficiency. In contrast, the T residue in the 5'-UTR from positions -1 to -5 was the least favourable nucleotide in translational efficiency. Furthermore, the effect of the sequence context in the -1 to -21 region of the 5'-UTR was conserved in different plant species. Based on these observations, we propose that the sequence context immediately upstream of the AUG initiation codon plays a crucial role in determining the translational efficiency of plant genes.

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.

Functional identification of sorting receptors involved in trafficking of soluble lytic vacuolar proteins in vegetative cells of Arabidopsis.

In eukaryotic cells, protein trafficking plays an essential role in biogenesis of proteins that belong to the endomembrane compartments. In this process, an important step is the sorting of organellar proteins depending on their final destinations. For vacuolar proteins, vacuolar sorting receptors (VSRs) and receptor homology-transmembrane-RING H2 domain proteins (RMRs) are thought to be responsible. Arabidopsis (Arabidopsis thaliana) contains seven VSRs. Among them, VSR1, VSR3, and VSR4 are involved in sorting storage proteins targeted to the protein storage vacuole (PSV) in seeds. However, the identity of VSRs for soluble proteins of the lytic vacuole in vegetative cells remains controversial. Here, we provide evidence that VSR1, VSR3, and VSR4 are involved in sorting soluble lytic vacuolar and PSV proteins in vegetative cells. In protoplasts from leaf tissues of vsr1vsr3 and vsr1vsr4 but not vsr5vsr6, and rmr1rmr2 and rmr3rmr4 double mutants, soluble lytic vacuolar (Arabidopsis aleurain-like protein:green fluorescent protein [GFP] and carboxypeptidase Y:GFP and PSV (phaseolin) proteins, but not the vacuolar membrane protein Arabidopsis ßFructosidase4:GFP, exhibited defects in their trafficking; they accumulated to the endoplasmic reticulum with an increased secretion into medium. The trafficking defects in vsr1vsr4 protoplasts were rescued by VSR1 or VSR4 but not VSR5 or AtRMR1. Furthermore, of the luminal domain swapping mutants between VSR1 and VSR5, the mutant with the luminal domain of VSR1, but not that of VSR5, rescued the trafficking defects of Arabidopsis aleurain-like protein:GFP and phaseolin in vsr1vsr4 protoplasts. Based on these results, we propose that VSR1, VSR3, and VSR4, but not other VSRs, are involved in sorting soluble lytic vacuolar and PSV proteins for their trafficking to the vacuoles in vegetative cells.

Identification of sorting motifs of AtßFruct4 for trafficking from the ER to the vacuole through the Golgi and PVC.

Although much is known about the molecular mechanisms involved in transporting soluble proteins to the central vacuole, the mechanisms governing the trafficking of membrane proteins remain largely unknown. In this study, we investigated the mechanism involved in targeting the membrane protein, AtßFructosidase 4 (AtßFruct4), to the central vacuole in protoplasts. AtßFruct4 as a green fluorescent protein (GFP) fusion protein was transported as a membrane protein during transit from the endoplasmic reticulum (ER) through the Golgi apparatus and the prevacuolar compartment (PVC). The N-terminal cytosolic domain of AtßFruct4 was sufficient for transport from the ER to the central vacuole and contained sequence motifs required for trafficking. The sequence motifs, LL and PI, were found to be critical for ER exit, while the EEE and LCPYTRL sequence motifs played roles in trafficking primarily from the trans Golgi network (TGN) to the PVC and from the PVC to the central vacuole, respectively. In addition, actin filaments and AtRabF2a, a Rab GTPase, played critical roles in vacuolar trafficking at the TGN and PVC, respectively. On the basis of these results, we propose that the vacuolar trafficking of AtßFruct4 depends on multiple sequence motifs located at the N-terminal cytoplasmic domain that function as exit and/or sorting signals in different stages during the trafficking process.

Efficacy of Plant-Made Human Recombinant ACE2 against COVID-19 in a Golden Syrian Hamster Model.

Coronavirus disease 2019 (COVID-19) is a novel infectious respiratory disease caused by SARS-CoV-2. We evaluated the efficacy of a plant-based human recombinant angiotensin-converting enzyme 2 (hrACE2) and hrACE2-foldon (hrACE2-Fd) protein against COVID-19. In addition, we analyzed the antiviral activity of hrACE2 and hrACE2-Fd against SARS-CoV-2 using real-time reverse-transcription PCR and plaque assays. The therapeutic efficacy was detected using the Golden Syrian hamster model infected with SARS-CoV-2. Both hrACE2 and hrACE2-Fd inhibited SARS-CoV-2 by 50% at concentrations below the maximum plasma concentration, with EC50 of 5.8 µg/mL and 6.2 µg/mL, respectively. The hrACE2 and hrACE2-Fd injection groups showed a tendency for decreased viral titers in nasal turbinate tissues on day 3 after virus inoculation; however, this decrease was not detectable in lung tissues. Histopathological examination on day 9 after virus inoculation showed continued inflammation in the SARS-CoV-2 infection group, whereas decreased inflammation was observed in both the hrACE2 and hrACE2-Fd injection groups. No significant changes were observed at other time points. In conclusion, the potential therapeutic efficacy of plant-based proteins, hrACE2 and hrACE2-Fd, against COVID-19 was confirmed in a SARS-CoV-2-inoculated Golden Syrian hamster model. Further preclinical studies on primates and humans are necessary to obtain additional evidence and determine the effectiveness of these therapies.

A Plant-Derived Maternal Vaccine against Porcine Epidemic Diarrhea Protects Piglets through Maternally Derived Immunity.

Newborn piglets are susceptible to a highly contagious enteritis caused by the porcine epidemic diarrhea virus (PEDV), associated with high levels of mortality worldwide. There is pressing need for a rapid, safe, and cost-effective vaccine to safeguard pigs from getting infected by PEDV. PEDV belongs to the coronavirus family and is characterized by high levels of mutability. The primary goal of a PEDV vaccine is to provide immunity to newborn piglets through vaccination of sows. Plant-based vaccines are becoming more popular because they have low manufacturing costs, are easily scalable, have high thermostability, and a long shelf life. This is in contrast to conventional vaccines which include inactivated, live, and/or recombinant types that can be expensive and have limited ability to respond to rapidly mutating viruses. The binding of the virus to host cell receptors is primarily facilitated by the N-terminal subunit of the viral spike protein (S1), which also contains several epitopes that are recognized by virus-neutralizing antibodies. As a result, we generated a recombinant S1 protein using a plant-based vaccine platform. We found that the recombinant protein was highly glycosylated, comparable to the native viral antigen. Vaccination of pregnant sows at four and two weeks before farrowing led to the development of humoral immunity specific to S1 in the suckling piglets. In addition, we noted significant viral neutralization titers in both vaccinated sows and piglets. When challenged with PEDV, piglets born from vaccinated sows displayed less severe clinical symptoms and significantly lower mortality rates compared to piglets born from non-vaccinated sows.

Plant-expressed Zika virus envelope protein elicited protective immunity against the Zika virus in immunocompetent mice.

Zika virus infection causes multiple clinical issues, including Guillain-Barré syndrome and neonatal malformation. Vaccination is considered as the only strategy for the prevention of ZIKV-induced clinical issues. This study developed a plant-based recombinant vaccine that transiently expressed the ZIKV envelope protein (ZikaEnv:aghFc) in Nicotiana benthamiana and evaluated the protective immunity afforded by it in immunocompetent mice. ZikaEnv:aghFc induced both humoral and cellular immunity at a low dose (1-5 µg). This immune-inducing potential was enhanced further when adjuvanted CIA09A. In addition, antigen-specific antibodies and neutralizing antibodies were vertically transferred from immunized females to their progeny and afforded both protective immunity to ZIKV and cross-protection to Dengue virus infection. These results suggest that our plant-based ZIKV vaccine provides a safe and efficient protective strategy with a competitive edge.

Plant produced endotoxin binding recombinant proteins effectively remove endotoxins from protein samples.

Lipopolysaccharides (LPS) are highly toxic compounds, even at a trace amount. When recombinant proteins are produced in E. coli, it is inevitable that LPS contaminates. However, LPS removal is still technically challenging and costly due to the high degree of solubility in a wide range of solvents. In this study, we explored the possibility of using the N-terminal region containing cysteine-rich, EGF-like, and sushi1-3 domains (CES3) of Factor C from the horseshoe crab Carcinoscorpius rotundicauda to develop a platform to remove LPS from recombinant proteins. We expressed CES3 as part of a recombinant protein, BiP:NT:CBM3:SUMO:CES3:His:HDEL, in Nicotiana benthamiana and found that purified or microcrystalline cellulose (MCC) bead-immobilised CES3 showed strong binding to LPS-containing E. coli. To produce CES3:CBM3 in an LPS-free environment, we generated Arabidopsis transgenic plants harbouring a recombinant gene, BiP:NT:SUMO:CES3:CBM3:HDEL, and found that transgenic plants mainly produce CES3:CBM3:His:HDEL, a truncated version of BiP:NT:SUMO:CES3:CBM3:HDEL via endogenous protease-mediated proteolytic processing in vivo. CES3:CBM3:HDEL purified from Arabidopsis plant extracts and immobilised onto MCC beads removed LPS contamination from protein samples. We propose that the CES3:CBM3 fusion protein produced in plants and immobilised on MCC beads can be a robust and easy platform for LPS removal from recombinant proteins.

Recombinant proteins of spike protein of SARS-CoV-2 with the Omicron receptor-binding domain induce production of highly Omicron-specific neutralizing antibodies.

Various vaccines have been developed to fight severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus responsible for the coronavirus disease 2019 pandemic. However, new variants of SARS-CoV-2 undermine the effort to fight SARS-CoV-2. Here, we produced S proteins harboring the receptor-binding domain (RBD) of the Omicron variant in plants. Plant-produced S proteins together with adjuvant CIA09A triggered strong immune responses in mice. Antibodies in serum inhibited interaction of recombinant human angiotensin-converting enzyme 2 with RBD of the Omicron variant, but not RBD of other variants. These results suggest that antibodies induced by RBD of the Omicron variant are highly specific for the Omicron RBD, but not for that of other variants.

A Plant-Produced Porcine Parvovirus 1-82 VP2 Subunit Vaccine Protects Pregnant Sows against Challenge with a Genetically Heterologous PPV1 Strain.

Porcine parvovirus (PPV) causes reproductive failure in sows, and vaccination remains the most effective means of preventing infection. The NADL-2 strain has been used as a vaccine for ~50 years; however, it does not protect animals against genetically heterologous PPV strains. Thus, new effective and safe vaccines are needed. In this study, we aimed to identify novel PPV1 strains, and to develop PPV1 subunit vaccines. We isolated and sequenced PPV1 VP2 genes from 926 pigs and identified ten PPV1 strains (belonging to Groups C, D and E). We selected the Group D PPV1-82 strain as a vaccine candidate because it was close to the highly pathogenic 27a strain. The PPV1-82 VP2 protein was produced in Nicotiana benthamiana. It formed virus-like particles and exhibited a 211 agglutination value. The PPV1-190313 strain (Group E), isolated from an aborted fetus, was used as the challenging strain because it was pathogenic. The unvaccinated sow miscarried at 8 days postchallenge, and mummified fetuses were all PPV1-positive. By contrast, pregnant sows vaccinated with PPV1-82 VP2 had 9-11 Log2 antibody titers and produced normal fetuses after PPV1-190313 challenge. These results suggest the PPV1-82 VP2 subunit vaccine protects pregnant sows against a genetically heterologous PPV1 strain by inducing neutralizing antibodies.

Homomeric interaction of AtVSR1 is essential for its function as a vacuolar sorting receptor.

Vacuolar sorting receptors, BP80/VSRs, play a critical role in vacuolar trafficking of soluble proteins in plant cells. However, the mechanism of action of BP80 is not well understood. Here, we investigate the action mechanism of AtVSR1, a member of BP80 proteins in Arabidopsis (Arabidopsis thaliana), in vacuolar trafficking. AtVSR1 exists as multiple forms, including a high molecular mass homomeric complex in vivo. Both the transmembrane and carboxyl-terminal cytoplasmic domains of AtVSR1 are necessary for the homomeric interaction. The carboxyl-terminal cytoplasmic domain contains specific sequence information, whereas the transmembrane domain has a structural role in the homomeric interaction. In protoplasts, an AtVSR1 mutant, C2A, that contained alanine substitution of the region involved in the homomeric interaction, was defective in trafficking to the prevacuolar compartment and localized primarily to the trans-Golgi network. In addition, overexpression of C2A, but not wild-type AtVSR1, inhibited trafficking of soluble proteins to the vacuole and caused their secretion into the medium. Furthermore, C2A:hemagglutinin in transgenic plants interfered with the homomeric interaction of endogenous AtVSR1 and inhibited vacuolar trafficking of sporamin:green fluorescent protein. These data suggest that homomeric interaction of AtVSR1 is critical for its function as a vacuolar sorting receptor.

Porcine circovirus 2 capsid protein produced in N. benthamiana forms virus-like particles that elicit production of virus-neutralizing antibodies in guinea pigs.

Porcine circovirus type 2 (PCV2) is a non-enveloped, icosahedral virus of the Circoviridae family, with a small, circular, single-stranded DNA genome. PCV2 infections cause substantial economic losses in the pig industry worldwide. Currently, commercially produced PCV2 vaccines are expensive, whereas plant-based expression systems can produce recombinant proteins at low cost for use as vaccines. In this study, recombinant PCV2 capsid protein (rCap) was transiently expressed in Nicotiana benthamiana and purified by metal affinity chromatography, with a yield of 102 mg from 1 kg plant leaves. Electron microscopy confirmed that purified rCap self-assembled into virus-like particles (VLPs) at neutral pH. It was shown to provoke a strong immune response in guinea pigs. The results indicate that plant systems can enable production of large amounts of proteins to serve as candidates for subunit vaccines.

Plant-Produced N-glycosylated Ag85A Exhibits Enhanced Vaccine Efficacy Against Mycobacterium tuberculosis HN878 Through Balanced Multifunctional Th1 T Cell Immunity.

Tuberculosis (TB) is one of the deadliest infectious diseases worldwide and is caused by Mycobacterium tuberculosis (Mtb). An effective vaccine to prevent TB is considered the most cost-effective measure for controlling this disease. Many different vaccine antigen (Ag) candidates, including well-known and newly identified Ags, have been evaluated in clinical and preclinical studies. In this study, we took advantage of a plant system of protein expression using Nicotiana benthamiana to produce N-glycosylated antigen 85A (G-Ag85A), which is one of the most well-characterized vaccine Ag candidates in the field of TB vaccines, and compared its immunogenicity and vaccine efficacy with those of nonglycosylated Ag85A (NG-Ag85A) produced with an Escherichia coli system. Notably, G-Ag85A induced a more robust IFN-γ response than NG-Ag85A, which indicated that G-Ag85A is well recognized by the host immune system during Mtb infection. We subsequently compared the vaccine potential of G-Ag85A and NG-Ag85A by evaluating their immunological features and substantial protection efficacies. Interestingly, G-Ag85A yielded moderately enhanced long-term protective efficacy, as measured in terms of bacterial burden and lung inflammation. Strikingly, G-Ag85A-immunized mice showed a more balanced proportion of multifunctional Th1-biased immune responses with sustained IFN-γ response than did NG-Ag85A-immunized mice. Collectively, plant-derived G-Ag85A could induce protective and balanced Th1 responses and confer long-term protection against a hypervirulent Mtb Beijing strain infection, which indicated that plant-produced G-Ag85A might provide an excellent example for the production of an Mtb subunit vaccine Ag and could be an effective platform for the development of anti-TB vaccines.

Proportional subcellular localization of Arabidopsis thaliana RabA1a.

Subcellular localization of trafficking proteins in a single cell affects the assembly of trafficking machinery between organelles and vesicles throughout the targeting pathway. RabGTPase is one of the regulators to direct specific targeting of cargo molecules depending on GDP/GTP bound status. We have recently determined the crystal structures of GDP-bound inactive and both GTP- and GppNHp-bound active forms of Arabidopsis RabA1a. It is notable that the switch regions of RabA1a exhibit conformational changes derived by GDP or GTP binding. However, it was not clear that where the GDP- or GTP-bound RabA1a is localized at the subcellular level in a cell. Here we demonstrate that the distinct proportion of subcellular localization of RabA1a depends on its site-specific mutation as the GDP- or GTP-bound form. RabA1a proteins located at the plasma membrane, endosomes, and cytosol. While the GDP-bound form of RabA1aS27N located more at endosomes than the plasma membrane compared to the proportions of RabA1a wild-type, and the GTP-bound RabA1aQ72L located mainly at the plasma membrane in comparison to RabA1a wild-type and RabA1aS27N. These distinct proportional localizations of RabA1a enable a cognate interaction between inactive/active RabA1 and effector molecules to direct specific targeting of its cargo molecules.

Studying Protein Import into Chloroplasts Using Protoplasts.

The chloroplast is an essential organelle that is responsible for various cellular processes in plants, such as photosynthesis and the production of many secondary metabolites and lipids. Chloroplasts require a large number of proteins for these various physiological processes. Over 95% of chloroplast proteins are nucleus-encoded and imported into chloroplasts from the cytosol after translation on cytosolic ribosomes. Thus, the proper import or targeting of these nucleus-encoded chloroplast proteins to chloroplasts is essential for the proper functioning of chloroplasts as well as the plant cell. Nucleus-encoded chloroplast proteins contain signal sequences for specific targeting to chloroplasts. Molecular machinery localized to the chloroplast or cytosol recognize these signals and carry out the import process. To investigate the mechanisms of protein import or targeting to chloroplasts in vivo, we developed a rapid, efficient protoplast-based method to analyze protein import into chloroplasts of Arabidopsis. In this method, we use protoplasts isolated from leaf tissues of Arabidopsis. Here, we provide a detailed protocol for using protoplasts to investigate the mechanism by which proteins are imported into chloroplasts.

Fusion of a highly N-glycosylated polypeptide increases the expression of ER-localized proteins in plants.

Plants represent promising systems for producing various recombinant proteins. One key area of focus for improving this technology is developing methods for producing recombinant proteins at high levels. Many methods have been developed to increase the transcript levels of recombinant genes. However, methods for increasing protein production involving steps downstream of transcription, including translation, have not been fully explored. Here, we investigated the effects of N-glycosylation on protein production and provide evidence that N-glycosylation greatly increases the expression levels of ER-targeted recombinant proteins. Fusion of the extracellular domain (M domain) of protein tyrosine phosphatase receptor type C (CD45), which contains four putative N-glycosylation sites to a model protein, leptin at the C-terminus, increased recombinant protein levels by 6.1 fold. This increase was specific to ER-targeted proteins and was dependent on N-glycosylation. Moreover, expression levels of leptin, leukemia inhibitory factor and GFP were also greatly increased by fusion of M domain at either the N or C-terminus. Furthermore, the increase in protein levels resulted from enhanced translation, but not transcription. Based on these results, we propose that fusing a small domain containing N-glycosylation sites to target proteins is a powerful technique for increasing the expression levels of recombinant proteins in plants.

Vacuolar Sorting Receptor-Mediated Trafficking of Soluble Vacuolar Proteins in Plant Cells.

Vacuoles are one of the most prominent organelles in plant cells, and they play various important roles, such as degradation of waste materials, storage of ions and metabolites, and maintaining turgor. During the past two decades, numerous advances have been made in understanding how proteins are specifically delivered to the vacuole. One of the most crucial steps in this process is specific sorting of soluble vacuolar proteins. Vacuolar sorting receptors (VSRs), which are type I membrane proteins, are involved in the sorting and packaging of soluble vacuolar proteins into transport vesicles with the help of various accessory proteins. To date, large amounts of data have led to the development of two different models describing VSR-mediated vacuolar trafficking that are radically different in multiple ways, particularly regarding the location of cargo binding to, and release from, the VSR and the types of carriers utilized. In this review, we summarize current literature aimed at elucidating VSR-mediated vacuolar trafficking and compare the two models with respect to the sorting signals of vacuolar proteins, as well as the molecular machinery involved in VSR-mediated vacuolar trafficking and its action mechanisms.

EpsinR2 interacts with clathrin, adaptor protein-3, AtVTI12, and phosphatidylinositol-3-phosphate. Implications for EpsinR2 function in protein trafficking in plant cells.

Members of the epsin family of proteins (epsins) are characterized by the presence of an epsin N-terminal homology (ENTH) domain. Epsins have been implicated in various protein-trafficking pathways in animal and yeast (Saccharomyces cerevisiae) cells. Plant cells also contain multiple epsin-related proteins. In Arabidopsis (Arabidopsis thaliana), EPSIN1 is involved in vacuolar trafficking of soluble proteins. In this study, we investigated the role of Arabidopsis EpsinR2 in protein trafficking in plant cells. EpsinR2 contains a highly conserved ENTH domain but a fairly divergent C-terminal sequence. We found that the N-terminal ENTH domain specifically binds to phosphatidylinositol-3-P in vitro and has a critical role in the targeting of EpsinR2. Upon transient expression in protoplasts, hemagglutinin epitope-tagged EpsinR2 was translocated primarily to a novel cellular compartment, while a minor portion localized to the Golgi complex. Protein-binding experiments showed that EpsinR2 interacts with clathrin, AtVTI12, and the Arabidopsis homologs of adaptor protein-3 delta-adaptin and adaptor protein-2 alpha-adaptin. Localization experiments revealed that hemagglutinin epitope-tagged EpsinR2 colocalizes primarily with delta-adaptin and partially colocalizes with clathrin and AtVTI12. Based on these findings, we propose that EpsinR2 plays an important role in protein trafficking through interactions with delta-adaptin, AtVTI12, clathrin, and phosphatidylinositol-3-P.