RESUMO
UDP-glucuronic acid (UDP-GlcA) is the precursor of many plant cell wall polysaccharides and is required for production of seed mucilage. Following synthesis in the cytosol, it is transported into the lumen of the Golgi apparatus, where it is converted to UDP-galacturonic acid (UDP-GalA), UDP-arabinose, and UDP-xylose. To identify the Golgi-localized UDP-GlcA transporter, we screened Arabidopsis thaliana mutants in genes coding for putative nucleotide sugar transporters for altered seed mucilage, a structure rich in the GalA-containing polysaccharide rhamnogalacturonan I. As a result, we identified UUAT1, which encodes a Golgi-localized protein that transports UDP-GlcA and UDP-GalA in vitro. The seed coat of uuat1 mutants had less GalA, rhamnose, and xylose in the soluble mucilage, and the distal cell walls had decreased arabinan content. Cell walls of other organs and cells had lower arabinose levels in roots and pollen tubes, but no differences were observed in GalA or xylose contents. Furthermore, the GlcA content of glucuronoxylan in the stem was not affected in the mutant. Interestingly, the degree of homogalacturonan methylation increased in uuat1 These results suggest that this UDP-GlcA transporter plays a key role defining the seed mucilage sugar composition and that its absence produces pleiotropic effects in this component of the plant extracellular matrix.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Complexo de Golgi/metabolismo , Proteínas de Transporte de Nucleotídeos/metabolismo , Polissacarídeos/metabolismo , Sementes/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Parede Celular/genética , Parede Celular/metabolismo , Regulação da Expressão Gênica de Plantas , Immunoblotting , Microscopia Confocal , Mutação , Proteínas de Transporte de Nucleotídeos/genética , Pectinas/metabolismo , Plantas Geneticamente Modificadas , Sementes/genética , Açúcares de Uridina Difosfato/metabolismoRESUMO
Upon imbibition, epidermal cells of Arabidopsis thaliana seeds release a mucilage formed mostly by pectic polysaccharides. The Arabidopsis mucilage is composed mainly of unbranched rhamnogalacturonan-I (RG-I), with low amounts of cellulose, homogalacturonan, and traces of xylan, xyloglucan, galactoglucomannan, and galactan. The pectin-rich composition of the mucilage and their simple extractability makes this structure a good candidate to study the biosynthesis of pectic polysaccharides and their modification. Here, we characterize the mucilage phenotype of a mutant in the UDP-rhamnose/galactose transporter 2 (URGT2), which exhibits a reduction in RG-I and also shows pleiotropic changes, suggesting the existence of compensation mechanisms triggered by the lack of URGT2. To gain an insight into the possible compensation mechanisms activated in the mutant, we performed a transcriptome analysis of developing seeds using RNA sequencing (RNA-seq). The results showed a significant misregulation of 3149 genes, 37 of them (out of the 75 genes described to date) encoding genes proposed to be involved in mucilage biosynthesis and/or its modification. The changes observed in urgt2 included the up-regulation of UAFT2, a UDP-arabinofuranose transporter, and UUAT3, a paralog of the UDP-uronic acid transporter UUAT1, suggesting that they play a role in mucilage biosynthesis. Mutants in both genes showed changes in mucilage composition and structure, confirming their participation in mucilage biosynthesis. Our results suggest that plants lacking a UDP-rhamnose/galactose transporter undergo important changes in gene expression, probably to compensate modifications in the plant cell wall due to the lack of a gene involved in its biosynthesis.
Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Proteínas de Transporte de Monossacarídeos/genética , Mucilagem Vegetal/biossíntese , Transcriptoma , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Proteínas de Transporte de Monossacarídeos/metabolismo , MutaçãoRESUMO
The cell wall is a complex extracellular matrix composed primarily of polysaccharides. Noncellulosic polysaccharides, glycoproteins and proteoglycans are synthesized in the Golgi apparatus by glycosyltransferases (GTs), which use nucleotide sugars as donors to glycosylate nascent glycan and glycoprotein acceptors that are subsequently exported to the extracellular space. Many nucleotide sugars are synthesized in the cytosol, leading to a topological issue because the active sites of most GTs are located in the Golgi lumen. Nucleotide sugar transporters (NSTs) overcome this problem by translocating nucleoside diphosphate sugars from the cytosol into the lumen of the organelle. The structures of the cell wall components synthesized in the Golgi are diverse and complex; therefore, transporter activities are necessary so that the nucleotide sugars can provide substrates for the GTs. In this review, we describe the topology of reactions involved in polysaccharide biosynthesis in the Golgi and focus on the roles of NSTs as well as their impacts on cell wall structure when they are altered.
Assuntos
Parede Celular/genética , Células Vegetais/metabolismo , Polissacarídeos/biossíntese , Açúcares/metabolismo , Transporte Biológico/genética , Parede Celular/química , Parede Celular/metabolismo , Glicosilação , Complexo de Golgi/genética , Complexo de Golgi/metabolismo , Proteínas de Transporte de Monossacarídeos , Nucleotídeos/química , Nucleotídeos/metabolismo , Polissacarídeos/genéticaRESUMO
ESCRT proteins mediate membrane remodeling and scission events and are essential for endosomal sorting of plasma membrane proteins for degradation. We have identified a novel, plant-specific ESCRT component called PROS (POSITIVE REGULATOR OF SKD1) in Arabidopsis thaliana. PROS has a strong positive effect on the in vitro ATPase activity of SKD1 (also known as Vacuolar Protein Sorting 4 or VPS4), a critical component required for ESCRT-III disassembly and endosomal vesiculation. PROS interacts with both SKD1 and the SKD1-positive regulator LIP5/VTA1. We have identified a putative MIM domain within PROS that mediate the interaction with the MIT domain of SKD1. Interestingly, whereas MIM domains are commonly found at the C terminus of ESCRT-III subunits, the PROS MIM domain is internal. The heterologous expression of PROS in yeast mutant cells lacking Vta1p partially rescues endosomal sorting defects. PROS is expressed in most tissues and cells types in Arabidopsis thaliana. Silencing of PROS leads to reduced cell expansion and abnormal organ growth.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/citologia , Arabidopsis/metabolismo , Proteínas de Transporte/metabolismo , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Desenvolvimento Vegetal , Adenosina Trifosfatases/metabolismo , Sequência de Aminoácidos , Arabidopsis/crescimento & desenvolvimento , Arabidopsis/ultraestrutura , Proteínas de Arabidopsis/química , Proteínas de Transporte/química , Proliferação de Células , Técnicas de Silenciamento de Genes , Inativação Gênica , Dados de Sequência Molecular , Corpos Multivesiculares/metabolismo , Mutação/genética , Folhas de Planta/citologia , Folhas de Planta/metabolismo , Folhas de Planta/ultraestrutura , Ligação Proteica , Estrutura Terciária de Proteína , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Especificidade da EspécieRESUMO
BACKGROUND: UDP-glucose: glycoprotein glucosyltransferase (UGGT) is a key player in the quality control mechanism (ER-QC) that newly synthesized glycoproteins undergo in the ER. It has been shown that the UGGT Arabidopsis orthologue is involved in ER-QC; however, its role in plant physiology remains unclear. RESULTS: Here, we show that two mutant alleles in the At1g71220 locus have none or reduced UGGT activity. In wild type plants, the AtUGGT transcript levels increased upon activation of the unfolded protein response (UPR). Interestingly, mutants in AtUGGT exhibited an endogenous up-regulation of genes that are UPR targets. In addition, mutants in AtUGGT showed a 30% reduction in the incorporation of UDP-Glucose into the ER suggesting that this enzyme drives the uptake of this substrate for the CNX/CRT cycle. Plants deficient in UGGT exhibited a delayed growth rate of the primary root and rosette as well as an alteration in the number of leaves. These mutants are more sensitive to pathogen attack as well as heat, salt, and UPR-inducing stressors. Additionally, the plants showed impairment in the establishment of systemic acquired resistance (SAR). CONCLUSIONS: These results show that a lack of UGGT activity alters plant vegetative development and impairs the response to several abiotic and biotic stresses. Moreover, our results uncover an unexpected role of UGGT in the incorporation of UDP-Glucose into the ER lumen in Arabidopsis thaliana.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimologia , Arabidopsis/fisiologia , Retículo Endoplasmático/metabolismo , Glucosiltransferases/metabolismo , Desenvolvimento Vegetal , Estresse Fisiológico , Adaptação Fisiológica/genética , Arabidopsis/genética , Arabidopsis/microbiologia , Estresse do Retículo Endoplasmático , Genes de Plantas , Mutação/genética , Frações Subcelulares/enzimologia , Resposta a Proteínas não DobradasRESUMO
The molecular mechanisms by which vascular tissues acquire their identities are largely unknown. Here, we report on the identification and characterization of VASCULATURE COMPLEXITY AND CONNECTIVITY (VCC), a member of a 15-member, plant-specific gene family in Arabidopsis (Arabidopsis thaliana) that encodes proteins of unknown function with four predicted transmembrane domains. Homozygous vcc mutants displayed cotyledon vein networks of reduced complexity and disconnected veins. Similar disconnections or gaps were observed in the provasculature of vcc embryos, indicating that defects in vein connectivity appear early in mutant embryo development. Consistently, the overexpression of VCC leads to an unusually high proportion of cotyledons with high-complexity vein networks. Neither auxin distribution nor the polar localization of the auxin efflux carrier were affected in vcc mutant embryos. Expression of VCC was detected in developing embryos and procambial, cambial, and vascular cells of cotyledons, leaves, roots, hypocotyls, and anthers. To evaluate possible genetic interactions with other genes that control vasculature patterning in embryos, we generated a double mutant for VCC and OCTOPUS (OPS). The vcc ops double mutant embryos showed a complete loss of high-complexity vascular networks in cotyledons and a drastic increase in both provascular and vascular disconnections. In addition, VCC and OPS interact physically, suggesting that VCC and OPS are part of a complex that controls cotyledon vascular complexity.
Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Genes de Plantas , Sementes/metabolismo , Sequência de Aminoácidos , Arabidopsis/embriologia , Arabidopsis/crescimento & desenvolvimento , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/fisiologia , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Homologia de Sequência de AminoácidosRESUMO
Zeins, the prolamin storage proteins found in maize (Zea mays), accumulate in accretions called protein bodies inside the endoplasmic reticulum (ER) of starchy endosperm cells. We found that genes encoding zeins, α-globulin, and legumin-1 are transcribed not only in the starchy endosperm but also in aleurone cells. Unlike the starchy endosperm, aleurone cells accumulate these storage proteins inside protein storage vacuoles (PSVs) instead of the ER. Aleurone PSVs contain zein-rich protein inclusions, a matrix, and a large system of intravacuolar membranes. After being assembled in the ER, zeins are delivered to the aleurone PSVs in atypical prevacuolar compartments that seem to arise at least partially by autophagy and consist of multilayered membranes and engulfed cytoplasmic material. The zein-containing prevacuolar compartments are neither surrounded by a double membrane nor decorated by AUTOPHAGY RELATED8 protein, suggesting that they are not typical autophagosomes. The PSV matrix contains glycoproteins that are trafficked through a Golgi-multivesicular body (MVB) pathway. MVBs likely fuse with the multilayered, autophagic compartments before merging with the PSV. The presence of similar PSVs also containing prolamins and large systems of intravacuolar membranes in wheat (Triticum aestivum) and barley (Hordeum vulgare) starchy endosperm suggests that this trafficking mechanism may be common among cereals.
Assuntos
Endosperma/metabolismo , Prolaminas/metabolismo , Vacúolos/metabolismo , Zea mays/metabolismo , Tomografia com Microscopia Eletrônica , Perfilação da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Complexo de Golgi/metabolismo , Microscopia Confocal , Prolaminas/genética , Transporte Proteico , Sementes/metabolismo , Zea mays/genética , Zeína/genética , Zeína/metabolismoRESUMO
Bisphenol A (BPA) is a chemical compound, considered as an "emerging pollutant", that appears ubiquitously, contaminating the environment and food. It is an endocrine disruptor, found in a multitude of consumer products, as it is a constituent of polycarbonate used in the manufacture of plastics and epoxy resins. Many studies have evaluated the effects of BPA, using a wide range of doses and animal models. In this work, we carried out a review of relevant research related to the effects of BPA on health, through studies performed at different doses, in different animal models, and in human monitoring studies. Numerous effects of BPA on health have been described; in different animal species, it has been reported that it interferes with fertility in both females and males and causes alterations in their offspring, as well as being associated with an increase in hormone-dependent pathologies. Similarly, exposure to BPA has been related to other diseases of great relevance in public health such as obesity, hypertension, diabetes, or neurodevelopmental disorders. Its ubiquity and nonmonotonic behavior, triggering effects at exposure levels considered "safe", make it especially relevant when both animal and human populations are constantly and inadvertently exposed to this compound. Its effects at low exposure levels make it essential to establish safe exposure levels, and research into the effects of BPA must continue and be focused from a "One Health" perspective to take into account all the factors that could intervene in the development of a disease in any exposed organism.
RESUMO
The molecular machinery orchestrating microautophagy, whereby eukaryotic cells sequester autophagic cargo by direct invagination of the vacuolar/lysosomal membrane, is still largely unknown, especially in plants. Here, we demonstrate microautophagy of storage proteins in the maize aleurone cells of the endosperm and analyzed proteins with potential regulatory roles in this process. Within the cereal endosperm, starchy endosperm cells accumulate storage proteins (mostly prolamins) and starch whereas the peripheral aleurone cells store oils, storage proteins, and specialized metabolites. Although both cell types synthesize prolamins, they employ different pathways for their subcellular trafficking. Starchy endosperm cells accumulate prolamins in protein bodies within the endoplasmic reticulum (ER), whereas aleurone cells deliver prolamins to vacuoles via an autophagic mechanism, which we show is by direct association of ER prolamin bodies with the tonoplast followed by engulfment via microautophagy. To identify candidate proteins regulating this process, we performed RNA-seq transcriptomic comparisons of aleurone and starchy endosperm tissues during seed development and proteomic analysis on tonoplast-enriched fractions of aleurone cells. From these datasets, we identified 10 candidate proteins with potential roles in membrane modification and/or microautophagy, including phospholipase-Dα5 and a possible EUL-like lectin. We found that both proteins increased the frequency of tonoplast invaginations when overexpressed in Arabidopsis leaf protoplasts and are highly enriched at the tonoplast surface surrounding ER protein bodies in maize aleurone cells, thus supporting their potential connections to microautophagy. Collectively, this candidate list now provides useful tools to study microautophagy in plants.
RESUMO
Bisphenol-A is an emerging pollutant that is widespread in the environment, and to which live beings are continuously and inadvertently exposed. It is a substance with an endocrine-disrupting capacity, causing alterations in the reproductive, immunological, and neurological systems, among others, as well as metabolic alterations. Our study aimed to assess its clinical signs, and effects on the most relevant blood biochemical parameters, and to evaluate pituitary and gonadal histology after a chronic exposure of adult mice to different BPA doses (0.5, 2, 4, 50 and 100 µg/kg BW/day) through their drinking water. The biochemical results showed that a marked significant reduction (p < 0.05) was produced in the levels of serum glucose, hypoproteinaemia and hypoalbuminemia in the groups exposed to the highest doses, whereas in the group exposed to 50 µg/kg BW/day the glucose and total protein levels dropped, and the animals exposed to 100 µg/kg BW/day experienced a diminution in albumin levels. In the case of the group exposed to 50 µg/kg BW/day, however, hypertriglyceridemia and hypercholesterolemia were determined, and the blood parameters indicating kidney alterations such as urea and creatinine experienced a significant increase (p < 0.05) with respect to the controls. Regarding the pituitary and gonads, none of the animals exposed presented histological alterations at the doses tested, giving similar images to those of the control group. These results suggest that continuous exposure to low BPA doses could trigger an inhibition of hepatic gluconeogenesis, which would result in a hypoglycaemic state, together with an induction of the enzymes responsible for lipidic synthesis, a mechanism by which the increase in the lipid and serum cholesterol levels could be explained. Likewise, the decline in the protein and albumin levels would be indicative of a possible hepatic alteration, and the increase in urea and creatinine would point to a possible renal perturbation, derived from continuous exposure to this xenobiotic. Based on our results, it could be said that chronic exposure to low BPA doses would not produce any clinical signs or histological pituitary-gonadal effects, but it could cause modifications in some blood biochemical parameters, that could initially indicate a possible hepatic and renal effect.
Assuntos
Disruptores Endócrinos , Gônadas , Camundongos , Animais , Creatinina , Relação Dose-Resposta a Droga , Glucose , Ureia , Albuminas , Disruptores Endócrinos/toxicidadeRESUMO
Bisphenol A (BPA) is considered as being an emerging pollutant, to which both animal and human populations are continuously and inadvertently exposed. The identification of indirect biomarkers of effect could be a key factor in determining early adverse outcomes from exposure to low doses of BPA. Thus, this study on mice aims to evaluate and identify indirect biomarkers of effect through the analysis of their blood biochemistry, and of certain reproduction parameters after exposure to different BPA concentrations (0.5, 2, 4, 50, and 100 µg/kg BW/day) in drinking water over generations. Our results showed that there were no modifications in the reproductive parameters evaluated, like estrous cycle duration, litter size, or the percentage of the young alive at reaching the weaning stage, at the exposure levels evaluated. However, there were modifications in the biochemical parameters, e.g., alterations in the glucose levels, that increased significantly (p < 0.05) in the breeders at the higher exposure doses (50 and 100 µg/kg BW/day in F1; 50 µg/kg BW/day in F2 and 100 µg/kg BW/day in F3), that would suggest that the BPA could induce hyperglycemia and its complications in adult animals, probably due to some damage in the pancreas cells; albumin, that increased in the breeders exposed to the highest dose in F1 and F3, inferring possible hepatic alterations. Further, total proteins showed a diminution in their values in F1 and F2, except the group exposed to 100 µg/kg BW/day, whereas in F3 the values of this parameter increased with respect to the control group, this aspect likely being related to a possible hepatic and renal alteration. Based on these results, glucose, albumin, and total proteins could initially be considered as early indicators of indirect effect after prolonged exposure to low BPA doses over generations.
RESUMO
Uridine 5'-diphosphate (UDP)-glucose is transported into the lumen of the endoplasmic reticulum (ER), and the Arabidopsis nucleotide sugar transporter AtUTr1 has been proposed to play a role in this process; however, different lines of evidence suggest that another transporter(s) may also be involved. Here we show that AtUTr3 is involved in the transport of UDP-glucose and is located at the ER but also at the Golgi. Insertional mutants in AtUTr3 showed no obvious phenotype. Biochemical analysis in both AtUTr1 and AtUTr3 mutants indicates that uptake of UDP-glucose into the ER is mostly driven by these two transporters. Interestingly, the expression of AtUTr3 is induced by stimuli that trigger the unfolded protein response (UPR), a phenomenon also observed for AtUTr1, suggesting that both AtUTr1 and AtUTr3 are involved in supplying UDP-glucose into the ER lumen when misfolded proteins are accumulated. Disruption of both AtUTr1 and AtUTr3 causes lethality. Genetic analysis showed that the atutr1 atutr3 combination was not transmitted by pollen and was poorly transmitted by the ovules. Cell biology analysis indicates that knocking out both genes leads to abnormalities in both male and female germ line development. These results show that the nucleotide sugar transporters AtUTr1 and AtUTr3 are required for the incorporation of UDP-glucose into the ER, are essential for pollen development and are needed for embryo sac progress in Arabidopsis thaliana.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Retículo Endoplasmático/metabolismo , Glucose/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Proteínas de Transporte de Nucleotídeos/metabolismo , Pólen/metabolismo , Difosfato de Uridina/metabolismo , Sequência de Aminoácidos , Arabidopsis/química , Arabidopsis/embriologia , Arabidopsis/genética , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Transporte Biológico , Genótipo , Complexo de Golgi/metabolismo , Proteínas de Membrana Transportadoras/química , Proteínas de Membrana Transportadoras/genética , Microscopia Eletrônica de Varredura , Dados de Sequência Molecular , Mutação , Proteínas de Transporte de Nucleotídeos/química , Proteínas de Transporte de Nucleotídeos/genética , Pólen/embriologia , Pólen/ultraestrutura , Alinhamento de Sequência , Homologia de Sequência de AminoácidosRESUMO
Developing maize (Zea mays) endosperms can be excised from the maternal tissues and undergo tissue/cell-type differentiation under in vitro conditions. We have developed a method to transform in vitro-grown endosperms using Agrobacterium tumefaciens and standard binary vectors. We show that both aleurone and starchy endosperm cells can be successfully transformed using a short cocultivation with A. tumefaciens cells. The highest transformation rates were obtained with the A. tumefaciens EHA101 strain and the pTF101.1 binary vector. The percentage of aleurone cells transformed following this method varied between 10% and 22% whereas up to the eighth layer of starchy endosperm cells underneath the aleurone layer showed transformed cells. Cultured endosperms undergo normal cell type (aleurone and starchy endosperm) differentiation and storage protein accumulation, making them suitable for cell biology and biochemical studies. In addition, transgenic cultured endosperms are able to express and accumulate epitope-tagged storage proteins that can be isolated for biochemical assays or used for immunolabeling techniques.
Assuntos
Agrobacterium tumefaciens/genética , Endosperma/genética , Transformação Genética , Zea mays/genética , Endosperma/citologia , Regulação da Expressão Gênica no Desenvolvimento , Regulação da Expressão Gênica de Plantas , Proteínas de Fluorescência Verde/genética , Microscopia de Fluorescência , Dados de Sequência Molecular , Plantas Geneticamente Modificadas/genética , Plasmídeos , Proteínas de Armazenamento de Sementes/metabolismoRESUMO
The synthesis of non-cellulosic polysaccharides occurs in the Golgi apparatus and requires a great number of metabolites (substrates and ions). Many enzymes use these substrates to add sugar residues to nascent polysaccharides chains or to introduce methyl and acetyl groups onto these polymers. Most of these metabolites are in the cytosol and their transport to the Golgi lumen is essential for proper polysaccharide biosynthesis. Different transporters activities have been described in Golgi membranes, but many more are thought to be present to provide all the substrates required for polysaccharide biosynthesis and to pump the ions for maintaining ionic homeostasis. Their functional analysis will help us to understand the role these transporters play in cell wall biosynthesis.
Assuntos
Parede Celular/metabolismo , Complexo de Golgi/metabolismo , Proteínas de Membrana Transportadoras/fisiologia , Polissacarídeos/biossíntese , Transporte Biológico , Biologia Computacional , Proteínas de Membrana Transportadoras/genética , Modelos Biológicos , Plantas/genética , Plantas/metabolismoRESUMO
Neuromuscular diseases represent a rare cause of dilated myocardiopathy, among them Duchenne muscular dystrophy is the most common. Transthoracic echocardiography and cardiac magnetic resonance imaging can assess cardiac involvement early. The case of a patient diagnosed with Duchenne muscular dystrophy who develops cardiac involvement during cardiology follow-up is presented below.
Assuntos
Humanos , Masculino , Adulto , Distrofia Muscular de Duchenne/diagnóstico , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/terapia , Cardiomiopatia Dilatada , Distrofina/genética , Distrofia Muscular de Duchenne/classificação , Distrofia Muscular de Duchenne/fisiopatologia , Diagnóstico Diferencial , Insuficiência CardíacaRESUMO
ESCRT (Endosomal Sorting Complex Required for Transport) proteins are required for the sorting of biosynthetic and endocytic proteins at multivesicular bodies (MVBs). Here, I describe an assay to evaluate conservation of endosomal sorting functions of plant ESCRT proteins by trans-species complementation analysis. The assay is based on the imaging of a fluorescent biosynthetic MVB cargo, the carboxypeptidase S (CPS) fused to the green fluorescent protein GFP, in yeast ESCRT mutants expressing putative plant orthologues of the missing yeast ESCRT components.
Assuntos
Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Endossomos/metabolismo , Biologia Molecular/métodos , Sequência de Aminoácidos , Complexos Endossomais de Distribuição Requeridos para Transporte/genética , Proteínas de Fluorescência Verde , Transporte Proteico/genética , Proteínas de Transporte Vesicular/metabolismoRESUMO
Endosomes regulate both the recycling and degradation of plasma membrane (PM) proteins, thereby modulating many cellular responses triggered at the cell surface. Endosomes also play a role in the biosynthetic pathway by taking proteins to the vacuole and recycling vacuolar cargo receptors. In plants, the trans-Golgi network (TGN) acts as an early/recycling endosome whereas prevacuolar compartments/multivesicular bodies (MVBs) take PM proteins to the vacuole for degradation. Recent studies have demonstrated that some of the molecular complexes that mediate endosomal trafficking, such as the retromer, the ADP-ribosylation factor (ARF) machinery, and the Endosomal Sorting Complexes Required for Transport (ESCRTs) have both conserved and specialized functions in plants. Whereas there is disagreement on the subcellular localization of the plant retromer, its function in recycling vacuolar sorting receptors (VSRs) and modulating the trafficking of PM proteins has been well established. Studies on Arabidopsis ESCRT components highlight the essential role of this complex in cytokinesis, plant development, and vacuolar organization. In addition, post-translational modifications of plant PM proteins, such as phosphorylation and ubiquitination, have been demonstrated to act as sorting signals for endosomal trafficking.
Assuntos
Endossomos/metabolismo , Plantas/metabolismo , Transporte Biológico , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Proteólise , Rede trans-Golgi/metabolismoRESUMO
Plasma membrane proteins internalized by endocytosis and targeted for degradation are sorted into lumenal vesicles of multivesicular bodies (MVBs) by the endosomal sorting complexes required for transport (ESCRT) machinery. Here, we show that the Arabidopsis thaliana ESCRT-related CHARGED MULTIVESICULAR BODY PROTEIN/CHROMATIN MODIFYING PROTEIN1A (CHMP1A) and CHMP1B proteins are essential for embryo and seedling development. Double homozygous chmp1a chmp1b mutant embryos showed limited polar differentiation and failed to establish bilateral symmetry. Mutant seedlings show disorganized apical meristems and rudimentary true leaves with clustered stomata and abnormal vein patterns. Mutant embryos failed to establish normal auxin gradients. Three proteins involved in auxin transport, PINFORMED1 (PIN1), PIN2, and AUXIN-RESISTANT1 (AUX1) mislocalized to the vacuolar membrane of the mutant. PIN1 was detected in MVB lumenal vesicles of control cells but remained in the limiting membrane of chmp1a chmp1b MVBs. The chmp1a chmp1b mutant forms significantly fewer MVB lumenal vesicles than the wild type. Furthermore, CHMP1A interacts in vitro with the ESCRT-related proteins At SKD1 and At LIP5. Thus, Arabidopsis CHMP1A and B are ESCRT-related proteins with conserved endosomal functions, and the auxin carriers PIN1, PIN2, and AUX1 are ESCRT cargo proteins in the MVB sorting pathway.
Assuntos
Arabidopsis/fisiologia , Proteínas de Transporte/metabolismo , Endocitose/fisiologia , Endossomos/metabolismo , Ácidos Indolacéticos/metabolismo , Sequência de Aminoácidos , Animais , Arabidopsis/citologia , Arabidopsis/embriologia , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Proteínas de Transporte/classificação , Proteínas de Transporte/genética , Complexos Endossomais de Distribuição Requeridos para Transporte , Teste de Complementação Genética , Humanos , Imuno-Histoquímica , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/metabolismo , Dados de Sequência Molecular , Fenótipo , Filogenia , Transporte Proteico , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Plântula/citologia , Plântula/fisiologia , Alinhamento de Sequência , Vacúolos/metabolismo , Proteínas de Transporte Vesicular/genética , Proteínas de Transporte Vesicular/metabolismoRESUMO
Reversible glycosylated polypeptides (RGPs) are highly conserved plant-specific proteins, which can perform self-glycosylation. These proteins have been shown essential in plants yet its precise function remains unknown. In order to understand the function of this self-glycosylating polypeptide, it is important to establish what factors are involved in the regulation of the RGP activity. Here we show that incubation at high ionic strength produced a high self-glycosylation level and a high glycosylation reversibility of RGP from Solanum tuberosum L. In contrast, incubation at low ionic strength led to a low level of glycosylation and a low glycosylation reversibility of RGP. The incubation at low ionic strength favored the formation of high molecular weight RGP-containing forms, whereas incubation at high ionic strength produced active RGP with a molecular weight similar to the one expected for the monomer. Our data also showed that glycosylation of RGP, in its monomeric form, was highly reversible, whereas, a low reversibility of the protein glycosylation was observed when RGP was part of high molecular weight structures. In addition, glycosylation of RGP increased the occurrence of non-monomeric RGP-containing forms, suggesting that glycosylation may favor multimer formation. Finally, our results indicated that RGP from Arabidopsis thaliana and Pisum sativum are associated to golgi membranes, as part of protein complexes. A model for the regulation of the RGP activity and its binding to golgi membranes based on the glycosylation of the protein is proposed where the sugars linked to oligomeric form of RGP in the golgi may be transferred to acceptors involved in polysaccharide biosynthesis.
Assuntos
Glicoproteínas/metabolismo , Proteínas de Plantas/metabolismo , Solanum tuberosum/metabolismo , Arabidopsis/metabolismo , Glicosilação , Complexo de Golgi/metabolismo , Membranas Intracelulares/metabolismo , Modelos BiológicosRESUMO
The folding of glycoproteins in the endoplasmic reticulum (ER) depends on a quality control mechanism mediated by the calnexin/calreticulin cycle. During this process, continuous glucose trimming and UDP-glucose-dependent re-glucosylation of unfolded glycoproteins takes place. To ensure proper folding, increases in misfolded proteins lead to up-regulation of the components involved in quality control through a process known as the unfolded protein response (UPR). Reglucosylation is catalyzed by the ER lumenal located enzyme UDP-glucose glycoprotein glucosyltransferase, but as UDP-glucose is synthesized in the cytosol, a UDP-glucose transporter is required in the calnexin/calreticulin cycle. Even though such a transporter has been hypothesized, no protein playing this role in the ER yet has been identified. Here we provide evidence that AtUTr1, a UDP-galactose/glucose transporter from Arabidopsis thaliana, responds to stimuli that trigger the UPR increasing its expression around 9-fold. The accumulation of AtUTr1 transcript is accompanied by an increase in the level of the AtUTr1 protein. Moreover, subcellular localization studies indicate that AtUTr1 is localized in the ER of plant cells. We reasoned that an impairment in AtUTr1 expression should perturb the calnexin/calreticulin cycle leading to an increase in misfolded protein and triggering the UPR. Toward that end, we analyzed an AtUTr1 insertional mutant and found an up-regulation of the ER chaperones BiP and calnexin, suggesting that these plants may be constitutively activating the UPR. Thus, we propose that in A. thaliana, AtUTr1 is the UDP-glucose transporter involved in quality control in the ER.