RÉSUMÉ
Mapping proteins in and associated with the Golgi apparatus reveals how this cellular compartment emerges in budding yeast and progresses over time.
Sujet(s)
Appareil de Golgi , SaccharomycetalesRÉSUMÉ
Transient gene expression in plant protoplasts facilitates the analysis of hybrid genes in a fast and reproducible manner. The technique is particularly powerful when studying basic conserved biochemical processes including de novo protein synthesis, modification, assembly, transport, and turnover. Unlike individual plants, protoplast suspensions can be divided into almost identical aliquots, allowing the analysis of independent variables with uncertainties restricted to minor pipetting errors/variations. Using the examples of protein secretion and ER retention, we describe the most advanced working practice of routinely preparing, electroporating, and analyzing Nicotiana benthamiana protoplasts. A single batch of electroporation-competent protoplasts permits up to 30 individual transfections. This is ideal to assess the influence of independent variables, such as point mutations, deletions or fusions, or the influence of a co-expressed effector gene in dose-response studies.
Sujet(s)
Nicotiana , Protoplastes , Nicotiana/génétique , Transport biologique , Transport des protéines , ÉlectroporationRÉSUMÉ
Accurately measuring the ability of the K/HDEL receptor (ERD2) to retain the ER cargo Amy-HDEL has questioned earlier results on which the popular receptor recycling model is based upon. Here we demonstrate that ERD2 Golgi-retention, rather than fast ER export supports its function. Ligand-induced ERD2 redistribution is only observed when the C-terminus is masked or mutated, compromising the signal that prevents Golgi-to-ER transport of the receptor. Forcing COPI mediated retrograde transport destroys receptor function, but introducing ER-to-Golgi export or cis-Golgi retention signals re-activate ERD2 when its endogenous Golgi-retention signal is masked or deleted. We propose that ERD2 remains fixed as a Golgi gatekeeper, capturing K/HDEL proteins when they arrive and releasing them again into a subdomain for retrograde transport back to the ER. An in vivo ligand:receptor ratio far greater than 100 to 1 strongly supports this model, and the underlying mechanism appears to be extremely conserved across kingdoms.
Sujet(s)
Protéines membranaires , Récepteurs peptidiques , Protéines membranaires/métabolisme , Ligands , Récepteurs peptidiques/génétique , Récepteurs peptidiques/métabolisme , Protéines de transport/métabolisme , Appareil de Golgi/métabolismeRÉSUMÉ
Accumulation of soluble proteins in the endoplasmic reticulum (ER) of plants is mediated by a receptor termed ER RETENTION DEFECTIVE2 (ERD2) or K/HDEL receptor. Using two gain-of-function assays and by complementing loss of function in Nicotiana benthamiana, we discovered that compromising the lumenal N terminus or the cytosolic C terminus with fluorescent fusions abolishes its biological function and profoundly affects its subcellular localization. Based on the confirmed asymmetrical topology of ERD2, we engineered a new fluorescent ERD2 fusion protein that retains biological activity. Using this fusion, we show that ERD2 is exclusively detected at the Golgi apparatus, unlike nonfunctional C-terminal fusions, which also label the ER. Moreover, ERD2 is confined to early Golgi compartments and does not show ligand-induced redistribution to the ER. We show that the cytosolic C terminus of ERD2 plays a crucial role in its function. Two conserved leucine residues that do not correspond to any known targeting motifs for ER-Golgi trafficking were shown to be essential for both ERD2 Golgi residency and its ability to mediate ER retention of soluble ligands. The results suggest that anterograde ER to Golgi transport of ERD2 is either extremely fast, well in excess of the bulk flow rate, or that ERD2 does not recycle in the way originally proposed.
Sujet(s)
Réticulum endoplasmique/métabolisme , Appareil de Golgi/métabolisme , Protéines membranaires/métabolisme , Nicotiana/métabolisme , Protéines végétales/métabolismeRÉSUMÉ
Due to the numerous roles plant vacuoles play in cell homeostasis, detoxification, and protein storage, the trafficking pathways to this organelle have been extensively studied. Recent evidence, however, suggests that our vision of transport to the vacuole is not as simple as previously imagined. Alternative routes have been identified and are being characterized. Intricate interconnections between routes seem to occur in various cases, complicating the interpretation of data. In this review, we aim to summarize the published evidence and link the emerging data with previous findings. We discuss the current state of information on alternative and classical trafficking routes to the plant vacuole.
Sujet(s)
Protéines végétales/métabolisme , Plantes/métabolisme , Voie de sécrétion , Vacuoles/métabolisme , Transport des protéinesRÉSUMÉ
Soluble hydrolases represent the main proteins of lysosomes and vacuoles and are essential to sustain the lytic properties of these organelles typical for the eukaryotic organisms. The sorting of these proteins from ER residents and secreted proteins is controlled by highly specific receptors to avoid mislocalization and subsequent cellular damage. After binding their soluble cargo in the early stage of the secretory pathway, receptors rely on their own sorting signals to reach their target organelles for ligand delivery, and to recycle back for a new round of cargo recognition. Although signals in cargo and receptor molecules have been studied in human, yeast and plant model systems, common denominators and specific examples of diversification have not been systematically explored. This review aims to fill this niche by comparing the structure and the function of lysosomal/vacuolar sorting receptors (VSRs) from these three organisms.
Sujet(s)
Protéines lysosomales membranaires/métabolisme , Lysosomes/métabolisme , Protéines de transport membranaire/métabolisme , Vacuoles/métabolisme , Protéines du transport vésiculaire/métabolisme , Animaux , Protéines fongiques/métabolisme , Protéines fongiques/physiologie , Humains , Protéines lysosomales membranaires/physiologie , Protéines de transport membranaire/physiologie , Protéines végétales/métabolisme , Protéines végétales/physiologie , Plantes/métabolisme , Conformation des protéines , Transport des protéines , Saccharomyces cerevisiae/métabolisme , Protéines du transport vésiculaire/physiologieRÉSUMÉ
Transport of proteins via the secretory pathway is controlled by a combination of signal dependent cargo selection as well as unspecific bulk flow of membranes and aqueous lumen. Using the plant vacuolar sorting receptor as model for membrane spanning proteins, we have distinguished bulk flow from signal mediated protein targeting in biosynthetic and endocytic transport routes and investigated the influence of transmembrane domain length. More specifically, long transmembrane domains seem to prevent ER retention, either by stimulating export or preventing recycling from post ER compartments. Long transmembrane domains also seem to prevent endocytic bulk flow from the plasma membrane, but the presence of specific endocytosis signals overrules this in a dominant manner.
Sujet(s)
Membrane cellulaire/métabolisme , Endocytose , Transduction du signal , Réticulum endoplasmique/métabolisme , Protéines mutantes/métabolisme , Transport des protéines , Délétion de séquence , Nicotiana/métabolisme , Vacuoles/métabolismeRÉSUMÉ
The cycling of vacuolar sorting receptors (VSRs) between early and late secretory pathway compartments is regulated by signals in the cytosolic tail, but the exact pathway is controversial. Here, we show that receptor targeting in tobacco (Nicotiana tabacum) initially involves a canonical coat protein complex II-dependent endoplasmic reticulum-to-Golgi bulk flow route and that VSR-ligand interactions in the cis-Golgi play an important role in vacuolar sorting. We also show that a conserved Glu is required but not sufficient for rate-limiting YXX-mediated receptor trafficking. Protein-protein interaction studies show that the VSR tail interacts with the µ-subunits of plant or mammalian clathrin adaptor complex AP1 and plant AP4 but not that of plant and mammalian AP2. Mutants causing a detour of full-length receptors via the cell surface invariantly cause the secretion of VSR ligands. Therefore, we propose that cycling via the plasma membrane is unlikely to play a role in biosynthetic vacuolar sorting under normal physiological conditions and that the conserved Ile-Met motif is mainly used to recover mistargeted receptors. This occurs via a fundamentally different pathway from the prevacuolar compartment that does not mediate recycling. The role of clathrin and clathrin-independent pathways in vacuolar targeting is discussed.
Sujet(s)
Appareil de Golgi/métabolisme , Nicotiana/métabolisme , Protéines végétales/métabolisme , Vacuoles/métabolisme , Séquence d'acides aminés , Réticulum endoplasmique/métabolisme , Données de séquences moléculaires , Protéines végétales/composition chimique , Liaison aux protéines , Transport des protéinesRÉSUMÉ
Here we examine the potential coupling between the synthesis of secretory proteins and the sensitivity of exocytosis to the concentration of free Ca(2+) in the cytosol ([Ca(2+)](i)) in plant cell. We therefore monitor in tobacco protoplasts the excursion of the membrane capacitance in response to an elevation of [Ca(2+)](i) as a measure for exocytotic activity. The data show that a ramp like elevation of [Ca(2+)](i) generates in protoplasts from wild type plants and from transgenic plants, which overexpress the secreted α-amylase, an exocytotic burst with an initial steep and a subsequent slow phase. The largest capacitive burst is obtained in α-amylase producing plants and the amplitude of the [Ca(2+)](i) evoked C(m) excursion is a function of the amylase synthesis of the plants. The data support a model according to which plant cells have at least two serial [Ca(2+)](i) sensitive processes in the final steps of their exocytotic pathway. The overproduction of a secreted cargo does not affect the kinetics of this process but the number of vesicles in pools upstream of the [Ca(2+)](i) sensitive steps.
Sujet(s)
Calcium/métabolisme , Exocytose/physiologie , Vésicules de sécrétion/métabolisme , Potentiels de membrane/physiologie , Feuilles de plante/enzymologie , Feuilles de plante/métabolisme , Protéines végétales/métabolisme , Végétaux génétiquement modifiés/métabolisme , Protoplastes/enzymologie , Protoplastes/métabolisme , Nicotiana/métabolisme , alpha-Amylases/biosynthèse , alpha-Amylases/métabolismeRÉSUMÉ
Delivery of proteins to the lytic vacuole in plants is a complex cascade of selective interactions that specifically excludes residents of the endoplasmic reticulum and secreted proteins. Vacuolar transport must be highly efficient to avoid mistargeting of hydrolytic enzymes to locations where they could be harmful. While plant vacuolar sorting signals have been well described for two decades, it is only during the last 5 years that a critical mass of data was gathered that begins to reveal how vacuolar sorting receptors (VSRs) may complete a full transport cycle. Yet, the field is far from reaching a consensus regarding the organelles that could be involved in vacuolar sorting, their potential biogenesis, and the ultimate recycling of membranes and protein machinery that maintain this pathway. This review will highlight the important landmarks in our understanding of VSR function and compare recent transport models that have been proposed so that an emerging picture of plant vacuolar sorting mechanisms can be drawn.
Sujet(s)
Protéines végétales/métabolisme , Vacuoles/métabolisme , Transport des protéines/physiologieRÉSUMÉ
Pentatricopeptide repeat (PPR) proteins belong to a family of approximately 450 members in Arabidopsis, of which few have been characterized. We identified loss of function alleles of SLO2, defective in a PPR protein belonging to the E+ subclass of the P-L-S subfamily. slo2 mutants are characterized by retarded leaf emergence, restricted root growth, and late flowering. This phenotype is enhanced in the absence of sucrose, suggesting a defect in energy metabolism. The slo2 growth retardation phenotypes are largely suppressed by supplying sugars or increasing light dosage or the concentration of CO2. The SLO2 protein is localized in mitochondria. We identified four RNA editing defects and reduced editing at three sites in slo2 mutants. The resulting amino acid changes occur in four mitochondrial proteins belonging to complex I of the electron transport chain. Both the abundance and activity of complex I are highly reduced in the slo2 mutants, as well as the abundance of complexes III and IV. Moreover, ATP, NAD+, and sugar contents were much lower in the mutants. In contrast, the abundance of alternative oxidase was significantly enhanced. We propose that SLO2 is required for carbon energy balance in Arabidopsis by maintaining the abundance and/or activity of complexes I, III, and IV of the mitochondrial electron transport chain.
Sujet(s)
Protéines d'Arabidopsis/métabolisme , Arabidopsis/métabolisme , Complexe I de la chaîne respiratoire/métabolisme , Métabolisme énergétique , Protéines mitochondriales/métabolisme , Édition des ARN , Adénosine triphosphate/métabolisme , Substitution d'acide aminé , Arabidopsis/génétique , Protéines d'Arabidopsis/génétique , Clonage moléculaire , Complexe I de la chaîne respiratoire/génétique , Régulation de l'expression des gènes végétaux , Mitochondries/métabolisme , Protéines mitochondriales/génétique , NAD/métabolisme , Phénotype , Saccharose/métabolismeRÉSUMÉ
Transient gene expression, in plant protoplasts or specific plant tissues, is a key technique in plant molecular cell biology, aimed at exploring gene products and their modifications to examine functional subdomains, their interactions with other biomolecules, and their subcellular localization. Here, we highlight some of the major advantages and potential pitfalls of the most commonly used transient gene expression models and illustrate how ectopic expression and the use of dominant mutants can provide insights into protein function.
Sujet(s)
Recherche , Voie de sécrétion , Transport biologique , Colorants fluorescents/métabolisme , Feuilles de plante/métabolisme , Feuilles de plante/microbiologie , Protoplastes/métabolisme , Protoplastes/ultrastructureRÉSUMÉ
GTPases of the Rab5 and Rab7 families were shown to control vacuolar sorting but their specific subcellular localization is controversial in plants. Here, we show that both the canonical as well as the plant-specific Rab5 reside at the newly discovered 'late prevacuolar compartment' (LPVC) while Rab7 partitions to the vacuolar membrane when expressed at low levels. Higher expression levels of wild-type Rab5 GTPases but not Rab7 lead to dose-dependent inhibition of biosynthetic vacuolar transport. In the case of Ara6, this included aberrant co-localization with markers for earlier post-Golgi compartments including the trans-Golgi network. However, nucleotide-free mutants of all three GTPases (Rha1, Ara6 and Rab7) cause stronger dose-dependent inhibition of vacuolar sorting. In addition, nucleotide-free Rha1 led to a later maturation defect and co-localization of markers for the prevacuolar compartment (PVC) and the LPVC. The corresponding Rab7 mutant strongly inhibited vacuolar delivery without merging of PVC and LPVC markers. Evidence for functional differentiation of the Rab5 family members is underlined by the fact that mutant Rha1 expression can be suppressed by increasing wild-type Rha1 levels while mutant Ara6 specifically titrates the nucleotide exchange factor Vps9. A model describing the sequential action of Rab5 and Rab7 GTPases is presented in the light of the current observations.
Sujet(s)
Nicotiana/métabolisme , Épiderme végétal/cytologie , Protéines végétales/métabolisme , Protoplastes/métabolisme , Vésicules de sécrétion/métabolisme , Protéines G rab/métabolisme , Cytosol/métabolisme , Appareil de Golgi/métabolisme , Facteurs d'échange de nucléotides guanyliques/métabolisme , Protéines luminescentes/génétique , Fusion membranaire/physiologie , Modèles biologiques , Mutation/physiologie , Protéines végétales/génétique , Protéines de fusion recombinantes/génétique , Protéines de fusion recombinantes/métabolisme , Nicotiana/cytologie , Nicotiana/génétique , Transfection/méthodes , Vacuoles/métabolisme , alpha-Amylases/métabolisme , Protéines G rab/génétique , Protéines Rab7 liant le GTP , Réseau trans-golgien/métabolismeRÉSUMÉ
We tested if different classes of vacuolar cargo reach the vacuole via distinct mechanisms by interference at multiple steps along the transport route. We show that nucleotide-free mutants of low molecular weight GTPases, including Rab11, the Rab5 members Rha1 and Ara6, and the tonoplast-resident Rab7, caused induced secretion of both lytic and storage vacuolar cargo. In situ analysis in leaf epidermis cells indicates a sequential action of Rab11, Rab5, and Rab7 GTPases. Compared with Rab5 members, mutant Rab11 mediates an early transport defect interfering with the arrival of cargo at prevacuoles, while mutant Rab7 inhibits the final delivery to the vacuole and increases cargo levels in prevacuoles. In contrast with soluble cargo, membrane cargo may follow different routes. Tonoplast targeting of an α-TIP chimera was impaired by nucleotide-free Rha1, Ara6, and Rab7 similar to soluble cargo. By contrast, the tail-anchored tonoplast SNARE Vam3 shares only the Rab7-mediated vacuolar deposition step. The most marked difference was observed for the calcineurin binding protein CBL6, which was insensitive to all Rab mutants tested. Unlike soluble cargo, α-TIP and Vam3, CBL6 transport to the vacuole was COPII independent. The results indicate that soluble vacuolar proteins follow a single route to vacuoles, while membrane spanning proteins may use at least three different transport mechanisms.
Sujet(s)
dGTPases/métabolisme , Nicotiana/métabolisme , Vacuoles/métabolisme , Agrobacterium/génétique , Agrobacterium/métabolisme , Protéines d'Arabidopsis/génétique , Protéines d'Arabidopsis/métabolisme , Transport biologique/physiologie , Vésicules COP/génétique , Vésicules COP/métabolisme , Protéines de liaison au calcium , Protéines de transport/génétique , Protéines de transport/métabolisme , dGTPases/génétique , Appareil de Golgi/métabolisme , Protéines membranaires/métabolisme , Épiderme végétal/enzymologie , Épiderme végétal/génétique , Épiderme végétal/métabolisme , Feuilles de plante/enzymologie , Feuilles de plante/génétique , Feuilles de plante/métabolisme , Feuilles de plante/microbiologie , Végétaux génétiquement modifiés , Protoplastes/métabolisme , Protéines de fusion recombinantes/génétique , Protéines de fusion recombinantes/métabolisme , Saccharomyces cerevisiae/génétique , Saccharomyces cerevisiae/métabolisme , Nicotiana/enzymologie , Nicotiana/microbiologie , Vacuoles/enzymologieRÉSUMÉ
Plant vacuolar sorting receptors (VSRs) display cytosolic Tyr motifs (YMPL) for clathrin-mediated anterograde transport to the prevacuolar compartment. Here, we show that the same motif is also required for VSR recycling. A Y612A point mutation in Arabidopsis thaliana VSR2 leads to a quantitative shift in VSR2 steady state levels from the prevacuolar compartment to the trans-Golgi network when expressed in Nicotiana tabacum. By contrast, the L615A mutant VSR2 leaks strongly to vacuoles and accumulates in a previously undiscovered compartment. The latter is shown to be distinct from the Golgi stacks, the trans-Golgi network, and the prevacuolar compartment but is characterized by high concentrations of soluble vacuolar cargo and the rab5 GTPase Rha1(RabF2a). The results suggest that the prevacuolar compartment matures by gradual receptor depletion, leading to the formation of a late prevacuolar compartment situated between the prevacuolar compartment and the vacuole.
Sujet(s)
Nicotiana/métabolisme , Vacuoles/métabolisme , Arabidopsis/génétique , Compartimentation cellulaire , Microscopie confocale , Microscopie électronique , Microscopie de fluorescence , Mutation ponctuelleRÉSUMÉ
Overexpression of the Golgi and endoplasmic reticulum (ER) syntaxins SYP31 and SYP81 strongly inhibits constitutive secretion. By comparing the secreted reporter alpha-amylase with the ER-retained reporter alpha-amylase-HDEL, it was concluded that SYP81 overexpression inhibits both retrograde and anterograde transport, while SYP31 overexpression mainly affected anterograde transport. Of the other interacting SNAREs investigated, only the overexpression of MEMB11 led to an inhibition of protein secretion. Although the position of a fluorescent tag does not influence the correct localization of the fusion protein, only N-terminal-tagged SYP31 retained the ability of the untagged SNARE to inhibit transport. C-terminal-tagged SYP31 failed to exhibit this effect. Overexpression of both wild-type and N-terminal-tagged syntaxins caused standard Golgi marker proteins to redistribute into the ER. Nevertheless, green fluorescent protein (GFP)-SYP31 was still visible as fluorescent punctae, which, unlike SYP31-GFP, were resistant to brefeldin A treatment. Immunogold electron microscopy showed that endogenous SYP81 is not only present at the ER but also in the cis Golgi, indicating that this syntaxin cycles between these two organelles. However, when expressed at non-inhibitory levels, YFP-SYP81 was seen to locate principally to subdomains of the ER. These punctate structures were physically separated from the Golgi, suggesting that they might possibly reflect the position of ER import sites.
Sujet(s)
Réticulum endoplasmique/métabolisme , Appareil de Golgi/métabolisme , Nicotiana/métabolisme , Protéines végétales/métabolisme , Protéines Qa-SNARE/métabolisme , Voie de sécrétion/physiologie , Clonage moléculaire , Réticulum endoplasmique/enzymologie , Réticulum endoplasmique/génétique , Réticulum endoplasmique/physiologie , Appareil de Golgi/enzymologie , Appareil de Golgi/génétique , Appareil de Golgi/physiologie , Protéines à fluorescence verte/métabolisme , Microscopie immunoélectronique , Feuilles de plante/enzymologie , Feuilles de plante/génétique , Feuilles de plante/métabolisme , Feuilles de plante/physiologie , Protéines végétales/biosynthèse , Protéines végétales/génétique , Plasmides , Transport des protéines/physiologie , Protoplastes/enzymologie , Protoplastes/métabolisme , Protéines Qa-SNARE/biosynthèse , Protéines Qa-SNARE/génétique , Nicotiana/enzymologie , Nicotiana/génétique , Nicotiana/physiologie , alpha-Amylases/métabolismeSujet(s)
Protéines d'Arabidopsis/métabolisme , Arabidopsis/métabolisme , Transport des protéines/physiologie , Arabidopsis/génétique , Protéines d'Arabidopsis/composition chimique , Protéines d'Arabidopsis/génétique , Protéines membranaires/métabolisme , Transport des protéines/génétique , Solubilité , Vacuoles/métabolismeRÉSUMÉ
Tomato spotted wilt virus (TSWV) particles are spherical and enveloped, an uncommon feature among plant infecting viruses. Previous studies have shown that virus particle formation involves the enwrapment of ribonucleoproteins with viral glycoprotein containing Golgi stacks. In this study, the localization and behaviour of the viral glycoproteins Gn and Gc were analysed, upon transient expression in plant protoplasts. When separately expressed, Gc was solely observed in the endoplasmic reticulum (ER), whereas Gn was found both within the ER and Golgi membranes. Upon co-expression, both glycoproteins were found at ER-export sites and ultimately at the Golgi complex, confirming the ability of Gn to rescue Gc from the ER, possibly due to heterodimerization. Interestingly, both Gc and Gn were shown to induce the deformation of ER and Golgi membranes, respectively, also observed upon co-expression of the two glycoproteins. The behaviour of both glycoproteins within the plant cell and the phenomenon of membrane deformation are discussed in light of the natural process of viral infection.
Sujet(s)
Réticulum endoplasmique/métabolisme , Glycoprotéines/métabolisme , Solanum lycopersicum/virologie , Tospovirus/pathogénicité , Protéines virales/métabolisme , Protéines bactériennes/génétique , Protéines bactériennes/métabolisme , Membrane cellulaire/métabolisme , Membrane cellulaire/ultrastructure , Réticulum endoplasmique/ultrastructure , Glycoprotéines/génétique , Appareil de Golgi/métabolisme , Appareil de Golgi/ultrastructure , Protéines luminescentes/génétique , Protéines luminescentes/métabolisme , Microscopie électronique à balayage , Précurseurs de protéines/génétique , Précurseurs de protéines/métabolisme , Protoplastes/ultrastructure , Protoplastes/virologie , Nicotiana/ultrastructure , Nicotiana/virologie , Tospovirus/métabolisme , Protéines virales/génétiqueRÉSUMÉ
The secretory pathway of eukaryotic cells comprises a network of organelles that connects three large membranes, the plasma membrane, the vacuole and the endoplasmic reticulum. The Golgi apparatus and the various post-Golgi organelles that control vacuolar sorting, secretion and endocytosis can be regarded as intermediate organelles of the endocytic and biosynthetic routes. Many processes in the secretory pathway have evolved differently in plants and cannot be studied using yeast or mammalian cells as models. The best characterized organelles are the Golgi apparatus and the prevacuolar compartment, but recent work has shed light on the role of the trans Golgi network, which has to be regarded as a separate organelle in plants. In this study, we wish to highlight recent findings regarding the late secretory pathway and its crosstalk with the early secretory pathway as well as the endocytic route in plants. Recently published findings and suggested models are discussed within the context of known features of the equivalent pathway in other eukaryotes.