RESUMEN
Vacuolar trafficking plays a vital role in plant growth and development. In this chapter, we describe a powerful technique for the evaluation of vacuolar protein trafficking, which is designated as GREEN FLUORESCENT SEED. Based on vacuole-targeted green fluorescent protein in Arabidopsis seeds, this method enables the nondestructive isolation of mutant seeds defective in vacuolar trafficking and their visual characterization.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Proteínas Fluorescentes Verdes/metabolismo , Semillas/metabolismo , Vacuolas/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Proteínas Fluorescentes Verdes/genética , Microscopía Confocal/métodos , Microscopía Fluorescente/métodos , Mutagénesis , Transporte de Proteínas , Semillas/genética , Vacuolas/genética , Proteínas de Transporte Vesicular/genéticaRESUMEN
Adaptor protein (AP) complexes play critical roles in protein sorting among different post-Golgi pathways by recognizing specific cargo protein motifs. Among the five AP complexes (AP-1-AP-5) in plants, AP-4 is one of the most poorly understood; the AP-4 components, AP-4 cargo motifs, and AP-4 functional mechanism are not known. Here, we identify the AP-4 components and show that the AP-4 complex regulates receptor-mediated vacuolar protein sorting by recognizing VACUOLAR SORTING RECEPTOR1 (VSR1), which was originally identified as a sorting receptor for seed storage proteins to target protein storage vacuoles in Arabidopsis (Arabidopsis thaliana). From the vacuolar sorting mutant library GREEN FLUORESCENT SEED (GFS), we isolated three gfs mutants that accumulate abnormally high levels of VSR1 in seeds and designated them as gfs4, gfs5, and gfs6. Their responsible genes encode three (AP4B, AP4M, and AP4S) of the four subunits of the AP-4 complex, respectively, and an Arabidopsis mutant (ap4e) lacking the fourth subunit, AP4E, also had the same phenotype. Mass spectrometry demonstrated that these four proteins form a complex in vivo. The four mutants showed defects in the vacuolar sorting of the major storage protein 12S globulins, indicating a role for the AP-4 complex in vacuolar protein transport. AP4M bound to the tyrosine-based motif of VSR1. AP4M localized at the trans-Golgi network (TGN) subdomain that is distinct from the AP-1-localized TGN subdomain. This study provides a novel function for the AP-4 complex in VSR1-mediated vacuolar protein sorting at the specialized domain of the TGN.
Asunto(s)
Complejo 4 de Proteína Adaptadora/metabolismo , Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Transporte de Proteínas/fisiología , Red trans-Golgi/metabolismo , Complejo 4 de Proteína Adaptadora/genética , Secuencias de Aminoácidos , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Mutación , Plantas Modificadas Genéticamente , Semillas/genética , Semillas/metabolismo , Vacuolas/metabolismoRESUMEN
Membrane trafficking to the protein storage vacuole (PSV) is a specialized process in seed plants. However, this trafficking mechanism to PSV is poorly understood. Here, we show that three types of Beige and Chediak-Higashi (BEACH)-domain proteins contribute to both vacuolar protein transport and effector-triggered immunity (ETI). We screened a green fluorescent seed (GFS) library of Arabidopsis mutants with defects in vesicle trafficking and isolated two allelic mutants gfs3 and gfs12 with a defect in seed protein transport to PSV. The gene responsible for the mutant phenotype was found to encode a putative protein belonging to group D of BEACH-domain proteins, which possess kinase domains. Disruption of other BEACH-encoding loci in the gfs12 mutant showed that BEACH homologs acted in a cascading manner for PSV trafficking. The epistatic genetic interactions observed among BEACH homologs were also found in the ETI responses of the gfs12 and gfs12 bchb-1 mutants, which showed elevated avirulent bacterial growth. The GFS12 kinase domain interacted specifically with the pleckstrin homology domain of BchC1. These results suggest that a cascade of multiple BEACH-domain proteins contributes to vacuolar protein transport and plant defense.
Asunto(s)
Proteínas de Arabidopsis/inmunología , Arabidopsis/metabolismo , Resistencia a la Enfermedad , Enfermedades de las Plantas/inmunología , Vacuolas/metabolismo , Arabidopsis/genética , Arabidopsis/inmunología , Arabidopsis/microbiología , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Enfermedades de las Plantas/microbiología , Estructura Terciaria de Proteína , Transporte de ProteínasRESUMEN
Flavonoids are the most important pigments for the coloration of flowers and seeds. In plant cells, flavonoids are synthesized by a multi-enzyme complex located on the cytosolic surface of the endoplasmic reticulum, and they accumulate in vacuoles. Two non-exclusive pathways have been proposed to mediate flavonoid transport to vacuoles: the membrane transporter-mediated pathway and the vesicle trafficking-mediated pathway. No molecules involved in the vesicle trafficking-mediated pathway have been identified, however. Here, we show that a membrane trafficking factor, GFS9, has a role in flavonoid accumulation in the vacuole. We screened a library of Arabidopsis thaliana mutants with defects in vesicle trafficking, and isolated the gfs9 mutant with abnormal pale tan-colored seeds caused by low flavonoid accumulation levels. gfs9 is allelic to the unidentified transparent testa mutant tt9. The responsible gene for these phenotypes encodes a previously uncharacterized protein containing a region that is conserved among eukaryotes. GFS9 is a peripheral membrane protein localized at the Golgi apparatus. GFS9 deficiency causes several membrane trafficking defects, including the mis-sorting of vacuolar proteins, vacuole fragmentation, the aggregation of enlarged vesicles, and the proliferation of autophagosome-like structures. These results suggest that GFS9 is required for vacuolar development through membrane fusion at vacuoles. Our findings introduce a concept that plants use GFS9-mediated membrane trafficking machinery for delivery of not only proteins but also phytochemicals, such as flavonoids, to vacuoles.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Flavonoides/metabolismo , Proteínas de la Membrana/metabolismo , Arabidopsis/fisiología , Arabidopsis/ultraestructura , Proteínas de Arabidopsis/genética , Transporte Biológico , Retículo Endoplásmico/metabolismo , Retículo Endoplásmico/ultraestructura , Flores/genética , Flores/fisiología , Flores/ultraestructura , Aparato de Golgi/metabolismo , Aparato de Golgi/ultraestructura , Membranas Intracelulares/metabolismo , Proteínas de la Membrana/genética , Fenotipo , Semillas/genética , Semillas/fisiología , Semillas/ultraestructura , Vacuolas/metabolismo , Vacuolas/ultraestructuraRESUMEN
The trans-Golgi network (TGN) is a tubular-vesicular organelle that matures from the trans cisternae of the Golgi apparatus. In plants, the TGN functions as a central hub for three trafficking pathways: the secretory pathway, the vacuolar trafficking pathway and the endocytic pathway. Here, we describe a novel TGN-localized membrane protein, CONTINUOUS VASCULAR RING (COV1), that is crucial for TGN function in Arabidopsis. The COV1 gene was originally identified from the stem vascular patterning mutant of Arabidopsis thaliana. However, the molecular function of COV1 was not identified. Fluorescently tagged COV1 proteins co-localized with the TGN marker proteins, SYNTAXIN OF PLANTS 4 (SYP4) and vacuolar-type H(+)-ATPase subunit a1 (VHA-a1). Consistently, COV1-localized compartments were sensitive to concanamycin A, a specific inhibitor of VHA. Intriguingly, cov1 mutants exhibited abnormal Golgi morphologies, including a reduction in the number of Golgi cisternae and a reduced association between the TGN and the Golgi apparatus. A deficiency in COV1 also resulted in a defect in vacuolar protein sorting, which was characterized by the abnormal accumulation of storage protein precursors in seeds. Moreover, we found that the development of an idioblast, the myrosin cell, was abnormally increased in cov1 leaves. Our results demonstrate that the novel TGN-localized protein COV1 is required for Golgi morphology, vacuolar trafficking and myrosin cell development.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Proteínas de la Membrana/metabolismo , Vacuolas/metabolismo , Red trans-Golgi/metabolismo , Diferenciación Celular/efectos de los fármacos , Concanavalina A/farmacología , Prueba de Complementación Genética , Proteínas Fluorescentes Verdes/metabolismo , Proteínas Luminiscentes/metabolismo , Fenotipo , Hojas de la Planta/efectos de los fármacos , Hojas de la Planta/metabolismo , Haz Vascular de Plantas/efectos de los fármacos , Haz Vascular de Plantas/metabolismo , Transporte de Proteínas/efectos de los fármacos , Proteínas de Almacenamiento de Semillas/metabolismo , Vacuolas/efectos de los fármacos , Red trans-Golgi/efectos de los fármacos , Red trans-Golgi/ultraestructura , Proteína Fluorescente RojaRESUMEN
Boron (B) is essential for plant growth but is toxic when present in excess. In the roots of Arabidopsis thaliana under B limitation, a boric acid channel, NIP5;1, and a boric acid/borate exporter, BOR1, are required for efficient B uptake and subsequent translocation into the xylem, respectively. However, under high-B conditions, BOR1 activity is repressed through endocytic degradation, presumably to avoid B toxicity. In this study, we investigated the localization of GFP-tagged NIP5;1 and BOR1 expressed under the control of their native promoters. Under B limitation, GFP-NIP5;1 and BOR1-GFP localized preferentially in outer (distal) and inner (proximal) plasma membrane domains, respectively, of various root cells. The polar localization of the boric acid channel and boric acid/borate exporter indicates the radial transport route of B toward the stele. Furthermore, mutational analysis revealed a requirement of tyrosine residues, in a probable cytoplasmic loop region of BOR1, for polar localization in various cells of the meristem and elongation zone. The same tyrosine residues were also required for vacuolar targeting upon high B supply. The present study of BOR1 and NIP5;1 demonstrates the importance of selective endocytic trafficking in polar localization and degradation of plant nutrient transporters for radial transport and homeostasis of plant mineral nutrients.
Asunto(s)
Antiportadores/metabolismo , Acuaporinas/metabolismo , Proteínas de Arabidopsis/metabolismo , Arabidopsis/citología , Arabidopsis/metabolismo , Boro/metabolismo , Polaridad Celular , Procesamiento Proteico-Postraduccional , Membrana Celular/metabolismo , Difusión , Proteínas Fluorescentes Verdes/metabolismo , Transporte de Proteínas , Proteínas Recombinantes de Fusión/metabolismo , Transducción de Señal , Tirosina/metabolismo , Vacuolas/metabolismoRESUMEN
Plants have developed their own defense strategies because they have no immune cells. A common plant defense strategy involves programmed cell death (PCD) at the infection site, but how the PCD-associated cell-autonomous immunity is executed in plants is not fully understood. Here we provide a novel mechanism underlying cell-autonomous immunity, which involves the fusion of membranes of a large central vacuole with the plasma membrane, resulting in the discharge of vacuolar antibacterial proteins to the outside of the cells, where bacteria proliferate. The extracellular fluid that was discharged from the vacuoles of infected leaves had both antibacterial activity and cell death-inducing activity. We found that a defect in proteasome function abolished the membrane fusion associated with both disease resistance and PCD in response to avirulent bacterial strains but not to a virulent strain. Furthermore, RNAi plants with a defective proteasome subunit PBA1 have reduced DEVDase activity, which is an activity associated with caspase-3, one of the executors of animal apoptosis. The plant counterpart of caspase-3 has not yet been identified. Our results suggest that PBA1 acts as a plant caspase-3-like enzyme. Thus, this novel defense strategy through proteasome-regulating membrane fusion of the vacuolar and plasma membranes provides plants with a mechanism for attacking intercellular bacterial pathogens.
Asunto(s)
Arabidopsis/microbiología , Membrana Celular/metabolismo , Fusión de Membrana , Enfermedades de las Plantas/microbiología , Pseudomonas syringae/fisiología , Vacuolas/metabolismo , Apoptosis , Arabidopsis/inmunología , Caspasa 1/metabolismo , Proteínas de Plantas/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismoRESUMEN
For mineral nutrients to be used by plants, they must be taken up from soil solutions into root cells and then transported to shoots. Mineral nutrient transporters play a central role in this process, and their expression and accumulation are known to be strictly regulated in response to change in nutrient conditions. Roots are cylindrically shaped organs with various types of cells. For the nutrients to move from soil solution toward the xylem they have to be transported across various types of cells. Nutrient condition-dependent accumulation and polar distributions of transporters in plant cells are established by membrane trafficking systems. The present article provides an overview of current findings regarding the membrane trafficking of mineral nutrient transporters and a discussion of future perspectives in this field of research.
Asunto(s)
Proteínas de Transporte de Membrana/metabolismo , Minerales/metabolismo , Proteínas de Plantas/metabolismo , Raíces de Plantas/metabolismo , Retículo Endoplásmico/metabolismo , Endosomas/metabolismo , Brotes de la Planta/metabolismo , Transporte de Proteínas , Suelo , Xilema/metabolismoRESUMEN
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.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis , Proteínas Fluorescentes Verdes/metabolismo , Transporte de Proteínas , Semillas/metabolismo , Vacuolas/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Antígenos de Plantas , Arabidopsis/citología , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Transporte Biológico , Globulinas/genética , Globulinas/metabolismo , Proteínas Fluorescentes Verdes/genética , Mutación , Señales de Clasificación de Proteína , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Proteínas de Almacenamiento de Semillas , Semillas/ultraestructura , Proteínas de Soja/genética , Proteínas de Soja/metabolismo , Vacuolas/química , Proteínas de Transporte Vesicular/genéticaRESUMEN
We isolated an Arabidopsis thaliana mutant, katamari2 (kam2), that has a defect in the organization of endomembranes. This mutant had deformed endosomes and formed abnormally large aggregates with various organelles. Map-based cloning revealed that kam2 is allelic to gravitropism defective 2 (grv2). The KAM2/GRV2 gene encodes a homolog of a DnaJ domain-containing RECEPTOR-MEDIATED ENDOCYTOSIS-8, which is considered to play a vital role in the endocytotic pathway from the plasma membrane to lysosomes in animal cells. Immunofluorescent staining showed that KAM2/GRV2 protein localizes on punctate structures, which did not merge with any markers for Golgi, trans-Golgi network, endosomes, or prevacuolar compartments. KAM2/GRV2, which does not have a predicted transmembrane domain, was peripherally associated with the membrane surface of uncharacterized compartments. KAM2/GRV2 was expressed at the early to middle stages of seed maturation. We found kam2 mis-sorted seed storage proteins by secreting them from cells, indicating that KAM2/GRV2 is involved in the transport of the proteins into protein storage vacuoles. kam2 had another defect in embryogenesis. Half of the developing kam2-1 cotyledons grew into the opposite space of the seeds before the walking stick-shaped embryo stage. Our findings suggest that KAM2/GRV2 is required for proper formation of the endosomes involving protein trafficking to the vacuoles and determination of growth axis of the embryo.
Asunto(s)
Proteínas de Arabidopsis/fisiología , Arabidopsis/metabolismo , Endosomas/fisiología , Semillas/metabolismo , Proteínas de Transporte Vesicular/fisiología , Arabidopsis/embriología , Arabidopsis/ultraestructura , Proteínas de Arabidopsis/análisis , Proteínas de Arabidopsis/genética , Clonación Molecular , Endosomas/metabolismo , Endosomas/ultraestructura , Proteínas Fluorescentes Verdes/análisis , Membranas Intracelulares/metabolismo , Mutación , Transporte de Proteínas/fisiología , Semillas/crecimiento & desarrollo , Semillas/ultraestructura , Vacuolas/metabolismo , Vacuolas/fisiología , Proteínas de Transporte Vesicular/análisis , Proteínas de Transporte Vesicular/genéticaRESUMEN
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.
Asunto(s)
Arabidopsis/ultraestructura , Endosomas/enzimología , Leucina/análogos & derivados , Nicotiana/ultraestructura , Péptido Hidrolasas/fisiología , Vacuolas/fisiología , Células Cultivadas , Inhibidores de Cisteína Proteinasa/farmacología , Endocitosis/fisiología , Endosomas/efectos de los fármacos , Leucina/farmacología , Fusión de Membrana/fisiología , Plantas Modificadas Genéticamente , Compuestos de Piridinio/farmacología , Compuestos de Amonio Cuaternario/farmacología , Quinacrina/farmacología , SacarosaRESUMEN
The seeds of higher plants accumulate large quantities of storage protein. During seed maturation, storage protein precursors synthesized on rough endoplasmic reticulum are sorted to protein storage vacuoles, where they are converted into the mature forms and accumulated. Previous attempts to determine the sorting machinery for storage proteins have not been successful. Here we show that a type I membrane protein, AtVSR1/AtELP, of Arabidopsis functions as a sorting receptor for storage proteins. The atvsr1 mutant missorts storage proteins by secreting them from cells, resulting in an enlarged and electron-dense extracellular space in the seeds. The atvsr1 seeds have distorted cells and smaller protein storage vacuoles than do WT seeds, and atvsr1 seeds abnormally accumulate the precursors of two major storage proteins, 12S globulin and 2S albumin, together with the mature forms of these proteins. AtVSR1 was found to bind to the C-terminal peptide of 12S globulin in a Ca2+-dependent manner. These findings demonstrate a receptor-mediated transport of seed storage proteins to protein storage vacuoles in higher plants.