Stubborn GFPs in Nicotiana tabacum vacuoles

Green fluorescent protein (GFP) allows the direct visualization of gene expression and sub cellular localization of fusion proteins in living cells. Many GFP variants have been developed to solve stability and emission problems. In this report the localization of different GFP fusion proteins, targeted to vacuoles, was studied in Nicotiana tabacum cv SR1. Even if a strong emission variant of the plant adapted GFP was used, no fluorescence was detected in differentiated tissues of N. tabacum with few exceptions. This model plant does not appear a good experimental system for the use of GFPs as vacuolar markers compared to Arabidopsis thaliana. In spite of this, our observations have evidenced a peculiar pattern of separated vacuoles in guard cells, providing new elements in the understanding of the vacuolar system organization.     

Identification of a vacuolar sucrose transporter in barley and Arabidopsis mesophyll cells by a tonoplast proteomic approach

The vacuole is the main cellular storage pool, where sucrose (Suc) accumulates to high concentrations. While a limited number of vacuolar membrane proteins, such as V-type H(+)-ATPases and H(+)-pyrophosphatases, are well characterized, the majority of vacuolar transporters are still unidentified, among them the transporter(s) responsible for vacuolar Suc uptake and release. In search of novel tonoplast transporters, we used a proteomic approach, analyzing the tonoplast fraction of highly purified mesophyll vacuoles of the crop plant barley (Hordeum vulgare). We identified 101 proteins, including 88 vacuolar and putative vacuolar proteins. The Suc transporter (SUT) HvSUT2 was discovered among the 40 vacuolar proteins, which were previously not reported in Arabidopsis (Arabidopsis thaliana) vacuolar proteomic studies. To confirm the tonoplast localization of this Suc transporter, we constructed and expressed green fluorescent protein (GFP) fusion proteins with HvSUT2 and its closest Arabidopsis homolog, AtSUT4. Transient expression of HvSUT2-GFP and AtSUT4-GFP in Arabidopsis leaves and onion (Allium cepa) epidermal cells resulted in green fluorescence at the tonoplast, indicating that these Suc transporters are indeed located at the vacuolar membrane. Using a microcapillary, we selected mesophyll protoplasts from a leaf protoplast preparation and demonstrated unequivocally that, in contrast to the companion cell-specific AtSUC2, HvSUT2 and AtSUT4 are expressed in mesophyll protoplasts, suggesting that HvSUT2 and AtSUT4 are involved in transport and vacuolar storage of photosynthetically derived Suc.     

Endosomal proteases facilitate the fusion of endosomes with vacuoles at the final step of the endocytotic pathway

The mechanism by which plasma membrane proteins are transported to vacuoles for degradation has not been well characterized in plants. To clarify how plasma membrane proteins are degraded, we monitored the endocytotic pathway in tobacco suspension-cultured BY-2 cells with a fluorescent endocytosis marker, FM4-64. Because of the efficient and rapid delivery of endosomes to the vacuoles, endosomes were scarcely detectable. Interestingly, we found that E-64d, an inhibitor of papain family proteases, caused the accumulation of a large number of endosomes in the cells under the sucrose-starved condition. This result indicates that E-64d attenuates the fusion of endosomes with vacuoles. We identified two papain homologues, which are localized in the endosomes, with a biotinylated inhibitor. We designated them as endosome-localized papains (ENPs). Immunofluorescent analysis revealed that vacuolar sorting receptor, a marker of prevacuolar compartment (PVC), was localized in the endosomes. This result and their acidic nature show that the endosomes correspond to PVC. These results suggest that ENPs facilitate the final step in the vacuolar trafficking pathway under the sucrose-starved condition. We further examined the effects of E-64d on two transgenic Arabidopsis plants that constitutively express a fusion protein composed of green fluorescent protein (GFP) and a plasma membrane protein (GFP-PIP2a or GFP-LTI6b). GFP fluorescence was observed on the plasma membrane of root cells in these transgenic plants. Treatment with E-64d induced the accumulation of GFP-fluorescent endosomes and inhibited the degradation of these fusion proteins. No GFP fluorescence was observed in vacuoles in E-64d-treated transgenic plants. Taken together, these results suggest that endosomal proteases are required for the fusion of endosomes with vacuoles at the final step in the endocytotic pathway for degradation of plasma membrane proteins in plants.     

Plant defensin AhPDF1.1 is not secreted in leaves but it accumulates in intracellular compartments

? Apart from their antifungal role, plant defensins have recently been shown to be involved in abiotic stress tolerance or in inhibition of root growth when added in plant culture medium. We studied the subcellular localization of these proteins, which may account for these different roles. ? Stable and transient expression of AhPDF1.1::GFP (green fluorescent protein) fusion proteins were analysed in yeast and plants. Functional tests established that the GFP tag did not alter the action of the defensin. Subcellular localization of AhPDF1.1 was characterized: by imaging AhPDF1.1::GFP together with organelle markers; and by immunolabelling AhPDF1.1 in Arabidopsis halleri and Arabidopsis thaliana leaves using a polyclonal serum. ? All our independent approaches demonstrated that AhPDF1.1 is retained in intracellular compartments on the way to the lytic vacuole, instead of being addressed to the apoplasm. ? These findings challenge the commonly accepted idea of secretion of defensins. The subcellular localization highlighted in this study could partly explain the dual role of plant defensins on plant cells and is of major importance to unravel the mechanisms of action of these proteins at the cellular level.     

Trafficking of phosphatidylinositol 3-phosphate from the trans-Golgi network to the lumen of the central vacuole in plant cells

Very limited information is available on the role of phosphatidylinositol 3-phosphate (PI[3]P) in vesicle trafficking in plant cells. To investigate the role of PI(3)P during the vesicle trafficking in plant cells, we exploited the PI(3)P-specific binding property of the endosome binding domain (EBD) (amino acids 1257 to 1411) of human early endosome antigen 1, which is involved in endosome fusion. When expressed transiently in Arabidopsis protoplasts, a green fluorescent protein (GFP):EBD fusion protein exhibited PI(3)P-dependent localization to various compartments--such as the trans-Golgi network, the prevacuolar compartment, the tonoplasts, and the vesicles in the vacuolar lumen--that varied with time. The internalized GFP:EBD eventually disappeared from the lumen. Deletion experiments revealed that the PI(3)P-dependent localization required the Rab5 binding motif in addition to the zinc finger motif. Overexpression of GFP:EBD inhibited vacuolar trafficking of sporamin but not trafficking of H(+)-ATPase to the plasma membrane. On the basis of these results, we propose that the trafficking of GFP:EBD reflects that of PI(3)P and that PI(3)P synthesized at the trans-Golgi network is transported to the vacuole through the prevacuolar compartment for degradation in plant cells.     

Novel type Arabidopsis thaliana H(+)-PPase is localized to the Golgi apparatus

Vacuolar H(+)-PPase, a membrane bound proton-translocating pyrophosphatase found in various species including plants, some protozoan and prokaryotes, has been demonstrated to be localized to the vacuolar membrane in plants. Using a GUS reporter system and a green fluorescent protein (GFP) fusion protein, we investigated the tissue distribution and the subcellular localization, respectively, of a novel type H(+)-PPase encoded by AVP2/AVPL1 identified in the Arabidopsis thaliana genome. We showed that AVP2/AVPL1 is highly expressed at the trichome and the filament of stamen. Furthermore, the fluorescence of GFP-tagged AVP2/AVPL1 showed small dot-like structures that were observed throughout the cytoplasm of various Arabidopsis cells under a fluorescent microscope. The distribution of this dot-like fluorescent pattern was apparently affected by a treatment with brefeldin A. Moreover, we demonstrated that most dot-like fluorescent structures colocalized with a Golgi resident protein. These findings suggest that this novel type H(+)-PPase resides on the Golgi apparatus rather than the vacuolar membrane.     

The Golgi-localized Arabidopsis endomembrane protein12 contains both endoplasmic reticulum export and Golgi retention signals at its C terminus

Endomembrane proteins (EMPs), belonging to the evolutionarily conserved transmembrane nine superfamily in yeast and mammalian cells, are characterized by the presence of a large lumenal N terminus, nine transmembrane domains, and a short cytoplasmic tail. The Arabidopsis thaliana genome contains 12 EMP members (EMP1 to EMP12), but little is known about their protein subcellular localization and function. Here, we studied the subcellular localization and targeting mechanism of EMP12 in Arabidopsis and demonstrated that (1) both endogenous EMP12 (detected by EMP12 antibodies) and green fluorescent protein (GFP)-EMP12 fusion localized to the Golgi apparatus in transgenic Arabidopsis plants; (2) GFP fusion at the C terminus of EMP12 caused mislocalization of EMP12-GFP to reach post-Golgi compartments and vacuoles for degradation in Arabidopsis cells; (3) the EMP12 cytoplasmic tail contained dual sorting signals (i.e., an endoplasmic reticulum export motif and a Golgi retention signal that interacted with COPII and COPI subunits, respectively); and (4) the Golgi retention motif of EMP12 retained several post-Golgi membrane proteins within the Golgi apparatus in gain-of-function analysis. These sorting signals are highly conserved in all plant EMP isoforms and, thus, likely represent a general mechanism for EMP targeting in plant cells.     

Localization of green fluorescent protein fusions with the seven Arabidopsis vacuolar sorting receptors to prevacuolar compartments in tobacco BY-2 cells

We have previously demonstrated that vacuolar sorting receptor (VSR) proteins are concentrated on prevacuolar compartments (PVCs) in plant cells. PVCs in tobacco (Nicotiana tabacum) BY-2 cells are multivesicular bodies (MVBs) as defined by VSR proteins and the BP-80 reporter, where the transmembrane domain (TMD) and cytoplasmic tail (CT) sequences of BP-80 are sufficient and specific for correct targeting of the reporter to PVCs. The genome of Arabidopsis (Arabidopsis thaliana) contains seven VSR proteins, but little is known about their individual subcellular localization and function. Here, we study the subcellular localization of the seven Arabidopsis VSR proteins (AtVSR1-7) based on the previously proven hypothesis that the TMD and CT sequences correctly target individual VSR to its final destination in transgenic tobacco BY-2 cells. Toward this goal, we have generated seven chimeric constructs containing signal peptide (sp) linked to green fluorescent protein (GFP) and TMD/CT sequences (sp-GFP-TMD/CT) of the seven individual AtVSR. Transgenic tobacco BY-2 cell lines expressing these seven sp-GFP-TMD-CT fusions all exhibited typical punctate signals colocalizing with VSR proteins by confocal immunofluorescence. In addition, wortmannin caused the GFP-marked prevacuolar organelles to form small vacuoles, and VSR antibodies labeled these enlarged MVBs in transgenic BY-2 cells. Wortmannin also caused VSR-marked PVCs to vacuolate in other cell types, including Arabidopsis, rice (Oryza sativa), pea (Pisum sativum), and mung bean (Vigna radiata). Therefore, the seven AtVSRs are localized to MVBs in tobacco BY-2 cells, and wortmannin-induced vacuolation of PVCs is a general response in plants.     

Mobilization of rubisco and stroma-localized fluorescent proteins of chloroplasts to the vacuole by an ATG gene-dependent autophagic process

During senescence and at times of stress, plants can mobilize needed nitrogen from chloroplasts in leaves to other organs. Much of the total leaf nitrogen is allocated to the most abundant plant protein, Rubisco. While bulk degradation of the cytosol and organelles in plants occurs by autophagy, the role of autophagy in the degradation of chloroplast proteins is still unclear. We have visualized the fate of Rubisco, stroma-targeted green fluorescent protein (GFP) and DsRed, and GFP-labeled Rubisco in order to investigate the involvement of autophagy in the mobilization of stromal proteins to the vacuole. Using immunoelectron microscopy, we previously demonstrated that Rubisco is released from the chloroplast into Rubisco-containing bodies (RCBs) in naturally senescent leaves. When leaves of transgenic Arabidopsis (Arabidopsis thaliana) plants expressing stroma-targeted fluorescent proteins were incubated with concanamycin A to inhibit vacuolar H(+)-ATPase activity, spherical bodies exhibiting GFP or DsRed fluorescence without chlorophyll fluorescence were observed in the vacuolar lumen. Double-labeled immunoelectron microscopy with anti-Rubisco and anti-GFP antibodies confirmed that the fluorescent bodies correspond to RCBs. RCBs could also be visualized using GFP-labeled Rubisco directly. RCBs were not observed in leaves of a T-DNA insertion mutant in ATG5, one of the essential genes for autophagy. Stroma-targeted DsRed and GFP-ATG8 fusion proteins were observed together in autophagic bodies in the vacuole. We conclude that Rubisco and stroma-targeted fluorescent proteins can be mobilized to the vacuole through an ATG gene-dependent autophagic process without prior chloroplast destruction.     

Searching for new cell wall protein genes in Arabidopsis

The objective of this study was to test an approach that combines bioinformatic and subcellular localization analysis to identify novel cell wall protein genes in Arabidopsis. Proteins with unknown function in the Arabidopsis genome were first identified and scanned for the presence of N-terminal signal peptides. The signal peptide-containing function-unknown proteins were further analyzed to eliminate the ones containing other sequences, such as endoplasmic reticulum and vacuole retention signals, that may prevent a protein from secretion into cell walls. The top ten genes passing the bioinformatic analysis were selected for protein subcellular localization using green fluorescence protein (GFP) as a reporter. A vector was constructed for high throughput gene-GFP fusion protein generation and overexpression in Arabidopsis for gene function analysis. Transformants of six genes showed reasonable expression of GFP fusion protein. However, none of the transformants showed GFP localization in cell walls. The low rate of new cell wall protein discovery suggests that the number of unidentified cell wall proteins in the Arabidopsis genome may be small.     

Rha1, an Arabidopsis Rab5 homolog,plays a critical role in the vacuolar trafficking of soluble cargo proteins

Rab proteins are members of the Ras superfamily of small GTP binding proteins and play important roles in various intracellular trafficking steps. We investigated the role of Rha1, an Arabidopsis Rab5 homolog, in intracellular trafficking in Arabidopsis protoplasts. In the presence of a dominant-negative mutant of Rha1, soluble vacuolar cargo proteins such as sporamin:green fluorescent protein (Spo:GFP) and Arabidopsis aleurain like protein:GFP are not delivered to the central vacuole; instead, they accumulate as a diffuse or punctate staining pattern within the cell. Spo:GFP at the punctate stains observed in the presence of hemagglutinin:Rha1[S24N] is colocalized with endogenous vacuolar sorting receptor (VSR(At-1)), which is known to localize primarily to the prevacuolar compartment, whereas Spo:GFP in the diffuse pattern is associated with tonoplasts. Furthermore, expression of Rha1[S24N] causes the secretion of a portion of the vacuolar proteins into medium. However, the inhibitory effect of Rha1[S24N] on vacuolar trafficking is relieved partially by coexpressed wild-type Rha1. Based on these results, we propose that Rha1 plays a critical role in the trafficking of soluble cargoes from the prevacuolar compartment to the central vacuole.     

Subcellular localization and targeting of glucocorticoid receptor protein fusions expressed in transgenic Arabidopsis thaliana

An animal system of inducible activation of protein fusions with the binding domain of glucocorticoid receptor (BDGR) was tested in Arabidopsis thaliana by monitoring dexamethasone (DEX)-induced nuclear targeting of reporter constructs. Two constructs containing green fluorescent protein (GFP), human homeobox protein Hanf-1 and Xenopus laevis BDGR were used, GFP/Hanf-1/BDGR and GFP/BDGR. The control construct contained GFP alone. In the absence of DEX both fusion proteins were uniformly distributed in the cytoplasm of root cells, but showed strong association with plastids in plant aerial parts. DEX treatment of roots prompted a strong and reversible nuclear accumulation of GFP/Hanf-1/BDGR, but not GFP/BDGR. Thus, in roots, the specific nuclear translocation of GFP/Hanf-1/BDGR was driven by Hanf-1 and tightly regulated by BDGR. However, in plant aerial parts treated with DEX, nuclear translocation of GFP/Hanf-1/BDGR was observed only in a few cases, and most part of the fusion protein was incorrectly and irreversibly targeted to plastids. Protease X digestion of isolated chloroplasts showed that BDGR fusion proteins were translocated into the chloroplast envelope and bound to envelope membranes, probably due to association with the chloroplast import apparatus. Thus, for efficient use of the glucocorticoid-inducible system in plants, it will be necessary to modify BDGR structure to prevent incorrect targeting of fusion proteins.     

N-terminal motifs in some plant disease resistance proteins function in membrane attachment and contribute to disease resistance

To investigate the role of N-terminal domains of plant disease resistance proteins in membrane targeting, the N termini of a number of Arabidopsis and flax disease resistance proteins were fused to green fluorescent protein (GFP) and the fusion proteins localized in planta using confocal microscopy. The N termini of the Arabidopsis RPP1-WsB and RPS5 resistance proteins and the PBS1 protein, which is required for RPS5 resistance, targeted GFP to the plasma membrane, and mutation of predicted myristoylation and potential palmitoylation sites resulted in a shift to nucleocytosolic localization. The N-terminal domain of the membrane-attached Arabidopsis RPS2 resistance protein was targeted incompletely to the plasma membrane. In contrast, the N-terminal domains of the Arabidopsis RPP1-WsA and flax L6 and M resistance proteins, which carry predicted signal anchors, were targeted to the endomembrane system, RPP1-WsA to the endoplasmic reticulum and the Golgi apparatus, L6 to the Golgi apparatus, and M to the tonoplast. Full-length L6 was also targeted to the Golgi apparatus. Site-directed mutagenesis of six nonconserved amino acid residues in the signal anchor domains of L6 and M was used to change the localization of the L6 N-terminal fusion protein to that of M and vice versa, showing that these residues control the targeting specificity of the signal anchor. Replacement of the signal anchor domain of L6 by that of M did not affect L6 protein accumulation or resistance against flax rust expressing AvrL567 but removal of the signal anchor domain reduced L6 protein accumulation and L6 resistance, suggesting that membrane attachment is required to stabilize the L6 protein.     

An ER-localized form of PV72, a seed-specific vacuolar sorting receptor, interferes the transport of an NPIR-containing proteinase in Arabidopsis leaves

Putative vacuolar sorting receptors that bind to the vacuolar targeting signals have been found in various plants; pumpkin PV72, pea BP-80 and Arabidopsis AtELP. PV72 is a seed-specific receptor that is predicted to sort seed storage proteins to protein storage vacuoles. Analysis by surface plasmon resonance showed that the lumenal domain of PV72 bound to an NPIR (a typical vacuolar targeting signal)-containing peptide of the precursor of a cysteine proteinase, AtALEU, in the presence of Ca(2+) (K(D) = 0.1 micro M). To elucidate the receptor-dependent transport of vacuolar proteins in plant cells, we produced transgenic Arabidopsis plants that expressed a fusion protein (PV72-HDEL) composed of the lumenal domain of PV72 and an endoplasmic reticulum (ER)-retention signal, HDEL. The expression of PV72-HDEL induced the accumulation of the AtALEU precursor. The accumulation level of the AtALEU precursor was dependent on that of PV72-HDEL. In contrast, it did not induce the accumulation of a precursor of another cysteine proteinase, RD21, which contains no NPIR. Detailed subcellular localization revealed that both the AtALEU precursor and PV72-HDEL accumulated in the ER fraction. We found that most of the AtALEU precursor molecules formed a complex with PV72-HDEL. The AtALEU precursor might be trapped by PV72-HDEL in the ER and not transported to the vacuoles. This in-planta analysis supports the hypothesis that an Arabidopsis homolog of PV72 functions as a sorting receptor for the NPIR-containing proteinase. The overall results suggest that vacuolar sorting receptors for the protein storage vacuoles and the lytic vacuoles share the similar recognition mechanism for a vacuolar targeting signal.     

Arabidopsis EPSIN1 plays an important role in vacuolar trafficking of soluble cargo proteins in plant cells via interactions with clathrin, AP-1, VTI11, and VSR1

Epsin and related proteins play important roles in various steps of protein trafficking in animal and yeast cells. Many epsin homologs have been identified in plant cells from analysis of genome sequences. However, their roles have not been elucidated. Here, we investigate the expression, localization, and biological role in protein trafficking of an epsin homolog, Arabidopsis thaliana EPSIN1, which is expressed in most tissues we examined. In the cell, one pool of EPSIN1 is associated with actin filaments, producing a network pattern, and a second pool localizes primarily to the Golgi complex with a minor portion to the prevacuolar compartment, producing a punctate staining pattern. Protein pull-down and coimmunoprecipitation experiments reveal that Arabidopsis EPSIN1 interacts with clathrin, VTI11, gamma-adaptin-related protein (gamma-ADR), and vacuolar sorting receptor1 (VSR1). In addition, EPSIN1 colocalizes with clathrin and VTI11. The epsin1 mutant, which has a T-DNA insertion in EPSIN1, displays a defect in the vacuolar trafficking of sporamin:green fluorescent protein (GFP), but not in the secretion of invertase:GFP into the medium. Stably expressed HA:EPSIN1 complements this trafficking defect. Based on these data, we propose that EPSIN1 plays an important role in the vacuolar trafficking of soluble proteins at the trans-Golgi network via its interaction with gamma-ADR, VTI11, VSR1, and clathrin.     

Why green fluorescent fusion proteins have not been observed in the vacuoles of higher plants

Green fluorescent protein (GFP) makes it possible for organelles and protein transport pathways to be visualized in living cells. However, GFP fluorescence has not yet been observed in the vacuoles of any organs of higher plants. We found that the fluorescence of a vacuole-targeted GFP was stably observed in the vacuoles of transgenic Arabidopsis plants under dark conditions, and that the fluorescence rapidly disappeared under light conditions. The vacuolar GFP was rapidly degraded within 1 h in the light, especially blue light. An inhibitor of vacuolar type H+-ATPase, concanamycin A, and an inhibitor of papain-type cysteine proteinase, E-64d, abolished both the light-dependent disappearance of GFP fluorescence and GFP degradation in the vacuoles. An in vitro assay showed that bacterially expressed GFP was degraded by extracts of Arabidopsis cultured-cell protoplasts at an acidic pH in the light. These results suggest that blue light induced a conformational change in GFP, and the resulting GFP in the vacuole was easily degraded by vacuolar papain-type cysteine proteinase(s) under the acidic pH. The light-dependent degradation accounts for the failure to observe GFP fluorescence in the vacuoles of plant organs. Our results show that stable GFP-fluoresced vacuoles are achieved by transferring the plants from the light into the dark before inspection with a fluorescent microscope. This might eliminate a large hurdle in studies of the vacuolar-targeting machinery and the organ- and stage-specific differentiation of endomembrane systems in plants.     

A complex and mobile structure forms a distinct subregion within the continuous vacuolar membrane in young cotyledons of Arabidopsis

The plant vacuole is a multifunctional organelle which is essential for growth and development. To visualize the dynamics of plant vacuolar membranes, gamma-TIP (tonoplast intrinsic protein) was fused to GFP and expressed in Arabidopsis thaliana. The marker molecule was targeted to the vacuolar membranes in most tissues, as expected. In rapidly expanding cells, some additional spherical structures were often observed within the lumen of vacuoles, which emitted strong fluorescence. To confirm their normal presence, we examined wild-type Arabidopsis cotyledons by transmission electron microscopy. The metal-contact rapid-freezing method revealed that the vacuolar lumen of epidermal cells contained many cytoplasmic projections, which often formed spherical structures (1-3 microm diameter) consisting of double membranes. Thus we concluded that these structures are authentic and named them 'bulbs'. Three-dimensional reconstruction from serial electron microscopic images demonstrates that bulbs are very intricately folded, but are continuous with the limiting vacuolar membrane. The fluorescence intensity of bulbs is about threefold higher than that of vacuolar membrane. GFP-AtRab75c, another marker of the vacuole, did not give fluorescent signals of bulbs in transgenic plants, but the existence of bulbs was still confirmed by electron microscopy. These results suggest that bulbs define a subregion in the continuous vacuolar membrane, where some proteins are concentrated and others segregated.     

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.     

Arabidopsis transportin1 is the nuclear import receptor for the circadian clock-regulated RNA-binding protein AtGRP7

We characterized the Arabidopsis orthologue of the human nuclear import receptor transportin1 (TRN1). Like the human receptor, Arabidopsis TRN1 recognizes nuclear import signals on proteins that are different from the classical basic nuclear localization signals. The M9 domain of human heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1) is the prototype of such signals. We show that AtTRN1 binds to similar domains in hnRNP-like proteins from plants. AtTRN1 also interacts with human hnRNP A1 and with yeast Nab2p, two classical import cargo proteins of transportin in these organisms. Like all nuclear transport receptors of the importin-beta family, AtTRN1 binds to the regulatory GTPase Ran from Arabidopsis. We demonstrated that the amino terminus of AtTRN1 is necessary for this interaction. Recombinant AtTRN1 conferred nuclear import of fluorescently labelled BSA-M9 peptide conjugates in permeabilized HeLa cells, functionally replacing human TRN1 in these in vitro nuclear import assays. We identified three plant substrate proteins that interact with AtTRN1 and contain M9-like domains: a novel Arabidopsis hnRNP that shows high similarity to human hnRNP A1 and two small RNA-binding proteins from Arabidopsis, AtGRP7 and AtGRP8. Nuclear import activity of the M9-like domains of these plant proteins was demonstrated in vivo by their ability to confer partial nuclear re-localisation of a GFP fusion protein containing a nuclear export signal. In addition, fluorescently labelled AtGRP7 was specifically imported into nuclei of permeabilized HeLa cells by Arabidopsis AtTRN1 and human TRN1. These results suggest that the transportin-mediated nuclear import pathway is highly conserved between man, yeast and plants.