Дисертації з теми "Biotin transporter"

Energy-Coupling-Factor (ECF)-Transporter sind Aufnahmesysteme für Vitamine und Übergangsmetallkationen in Prokaryoten. Sie bestehen aus den zwei unverwandten Membranproteinen S und T sowie einem Paar ABC-ATPasen (A). Die S-Einheit vermittelt die Substratspezifität. Die Kombination aus der T- und den A-Einheiten wird als ECF bezeichnet. In dieser Arbeit wurden Fragen zur kontrovers diskutierten Stöchiometrie der Untereinheiten von ECF-Transportern sowie zur zuvor postulierten Substrattransport-Funktion einzelner S-Komponenten auch ohne ECF untersucht. Dazu wurden der ECF-Biotintransporter BioMNY, mehrere natürlicherweise in Organismen ohne ECF existierende biotinspezifische S Einheiten (BioY) sowie zwei Vertreter der metallspezifischen ECF-Systeme genutzt. Die S-Einheit BioY des dreiteiligen Biotinimporters lag in vitro als Monomer und Dimer vor. Oligomeres BioY wurde außerdem in lebenden Bakterienzellen beobachtet. „Pull-down“-Experimente zeigten, dass die T Komponente BioN im BioMNY-Komplex zum Teil als Dimer vorlag. Wachstumsuntersuchungen bestätigten die Transportfunktion von acht solitär vorkommenden BioY. Die in vitro auch für diese BioY-Proteine nachgewiesene Dimerisierung könnte die Transportfunktion von BioY ohne ECF erklären. Die metallspezifischen S Einheiten CbiM/NikM interagieren mit für die Transportfunktion essentiellen, zusätzlichen Transmembranproteinen (N) und zeichnen sich durch eine Topologie mit sieben Transmembranhelices und einem extrem konservierten, weit in das Proteininnere hineinragenden N-Terminus aus. Die Metallbindestelle besteht aus vier Stickstoffatomen von Met1, His2 und His67 und wird durch ein Netz aus Wasserstoffbrückenbindungen stabilisiert. Die Transport¬funktion von CbiMN bzw. Nik(MN) ohne ECF wurde in vivo mittels des nickelabhängigen Enzyms Urease als Indikator für die intrazelluläre Nickelkonzentration verifiziert. Zum gegenwärtigen Zeitpunkt ist die Funktion der für den Transport essentiellen N-Komponente jedoch noch unklar.
Energy-coupling factor (ECF) transporters are uptake systems for vitamins and transition metal cations in prokaryotes. They consist of the two unrelated membrane proteins S and T, and a pair of ABC ATPases (A). The S unit mediates substrate specificity. The combination of the T and the A units is called ECF. In this thesis the controversially discussed stoichiometry of the subunits of ECF transporters and the postulated substrate transport function of solitary S units without ECF were analysed. For this purpose, the biotin-specific ECF transporter BioMNY, several biotin-specific S units (BioY) encoded in organisms lacking any recognizable ECF and two metal-specific ECF transporters were used. The S unit BioY of the tripartite biotin importer existed in vitro as monomer and dimer. Furthermore, oligomeric BioY was observed in living bacterial cells. Oligomerisation of a part of the T unit BioN in the BioMNY complex was shown by “pull-down”- experiments. Growth analyses confirmed the transport function of eight solitary BioY proteins. The dimerisation, also proved for these solitary BioY proteins in vitro, could be an explanation for the transport function of BioY without ECF. The metal-specific S units CbiM/NikM interact with additional and for the transport function essential transmembrane proteins (N). The S units consist of seven transmembrane helices and an extremely conserved N-terminus, which extends deeply into the protein. The metal-binding site consists of four nitrogen atoms from Met1, His2 and His67 and is stabilised by a series of hydrogen bonds. The transport function of CbiMN and Nik(MN) without ECF was verified respectively in vivo using the nickel-depending enzyme urease as an indicator for intracellular nickel concentration, respectively. However, the role of the N component, which is essential for transport activity, is currently under investigation.

The human glucose transporter GLUT1 is abundant in red blood cells, the blood-brain barrier and epithelial cells, where it mediates the transport of the energy metabolite, glucose. In the present work some properties of GLUT1, including affinity binding of both substrates and inhibitors, transport rates as well as permeabilities of aromatic amino acids and drug-membrane interactions were analyzed by chromatographic methods.

Reconstitution by size-exclusion chromatography on Superdex 75 from a detergent with a low CMC that provides monomeric GLUT1 was examined regarding D-glucose- and CB binding as well as D-glucose transport. Upon steric immobilization in Superdex 200 gel beads, residual detergent could be washed away and dissociation constants in the same range as reported for binding to GLUT1 reconstituted from other detergents were obtained. The transport rate into the GLUT1 proteoliposomes was low, probably due to residual detergent. Binding to GLUT1 at different pH was analyzed and the affinity of glucose and GLUT1 inhibitors was found to decrease with increasing pH (5–8.7). The average number of cytochalasin B-binding sites per GLUT1 monomers was, in most cases, approximately 0.4. GLUT1 may work as a functional monomer, dimer or oligomer. To determine whether GLUT1 was responsible for the transport of the aromatic amino acids tyrosine and tryptophan, uptake values and permeabilities of these amino acids into liposomes and GLUT1 proteoliposomes were compared to the permeabilities of D- and L- glucose in the same systems. Dihydrocytochalasin B was identified to be a new inhibitor of tyrosine and tryptophan transport into red blood cells. Ethanol turned out to inhibit the specific binding between CB and GLUT1 and also to decrease the partitioning of CB and drugs into lipid bilayers. A capacity factor for drug partitioning into membranes that allows comparison between columns with different amount of immobilized lipids was validated, and turned out to be independent of flow rate, amount of lipids and drug concentration in the ranges tested.

Membranproteine spielen eine wichtige Rolle in vielen biochemischen Prozessen der Zelle, wie zum Beispiel der Signaltransduktion, der Zelladhesion oder auch der Erkennung von Krankheitserregern. Viele dieser Proteine sind von Bedeutung für die Entwicklung neuer innovativer Medikamente. Somit hat auch die Entwicklung von Sensoren, die die Untersuchung von Membranproteinen in ihrer natürlichen Umgebung erlauben an Bedeutung gewonnen [1]. Thema dieser Doktorarbeit war die Entwicklung von Analysekonzepten die es ermöglichen unterschiedliche Aspekte von Membraninteraktionen zu untersuchen und zu quantifizieren. Als Analysemethode wurde dafür reflektometrische Interferenz Spektroskopie (RIfS) eine markierungsfreie, optische Methode verwendet. RIfS erlaubt es die Höhe dünner transparenter Filme zu bestimmen, indem das Weißlicht-Reflexionspektrum eines solchen Films aufgezeichnet wird. Durch die Überlagerung der in dem Film mehrfach reflektierten Teilstrahlen entsteht ein Interferenzmuster im Reflexionsspektrum, welches Aufschluß gibt über die Schichtdicke und den Brechungsindex des transparenten Films. Es wurde bereits gezeigt, dass RIfS eine geeignete Methode zur Untersuchung von Protein-ProteinWechselwirkungen ist [2]. Aus diesem Grund wurde RIfS als Detektionsverfahren für die Entwicklung eines Membransensors gewählt. Im Laufe dieser Arbeit entstanden zwei Aufbauten für reflektometrische Messungen. Ein Standard RIfS Aufbau und ein Instrument das die Methode mit Fluoreszenz-Mikroskopie kombiniert. Um dieWechselwirkung von Proteinen selbst und Proteinen mit Membranbestandteilen wie Lipiden zu untersuchen, wurde ein Konzept basierend auf festkörperunterstützten Membranen entwickelt. Dieses Experiment erlaubt es die Wechselwirkungen auf artifiziellen Membranen, sowie auf rekonstituierten Zellmembranen zu untersuchen. Zudem wurde ein Analysekonzept mit Nano-BLMs entwickelt, dass es erlaubt den simultanen Transport von Molekülen in ein membranverschlossenes Kompartiment hinein als auch heraus zu beobachten. Neben diesen membranbasierten Experimenten wurde auch ein Konzept entwickelt, welches es erlaubt die molekulare Erkennungsreaktion von sehr kleiner Analyten direkt zu messen. Dieses Messkonzept erlaubt es die Bindung von Molekülen mit sehr kleinem Molekulargewicht an einen auf dem Sensor immobilisierten Partner direkt zu quantifizieren.

Une importante avancée dans la compréhension des mécanismes gouvernant le processus de transport des ions iodures à l’intérieur des cellules thyroïdiennes a été le clonage en 1996 de la protéine responsable de ce transport : le symporteur Na/I (ou NIS). De nombreuses recherches ont été conduites depuis afin de caractériser cette protéine ainsi que les mécanismes qui régulent son expression et son activité. Les mécanismes cellulaires de régulation du transport et les protéines impliquées dans la régulation post-traductionnelle du symporteur restent toutefois largement inconnus. La compréhension de l’ensemble de ces mécanismes permettrait pourtant d’améliorer le traitement d’un grand nombre de patients. Le transport d’iode est en effet non seulement impliqué dans différentes pathologies de la thyroïde, mais aussi dans les contaminations à l’iode radioactif consécutives aux accidents nucléaires et dans de prometteuses stratégies de thérapie génique anticancéreuses. La chémogénomique, aussi appelée génétique chimique, est une approche multidisciplinaire dont le but est d’explorer les systèmes vivants au moyen de petites molécules organiques. Afin de mieux comprendre les mécanismes qui gouvernent le transport d’iode, notre laboratoire a mis en place une stratégie de génétique chimique qui a permis dans un premier temps de découvrir 10 molécules capables d’inhiber le transport d’iode. L’objectif de cette thèse était d’identifier les cibles protéiques de deux de ces molécules : ITB5 et ITB2. Des études d’électrophysiologie et de flux isotopique ayant montré que ces deux molécules ont un mode d’action différent, leur étude devait permettre d’identifier au moins deux protéines impliquées dans le transport des ions iodures.Afin d’identifier les protéines cibles d’ITB5 et d’ITB2, des sondes ont été synthétisées. Ces sondes sont constituées du composé d’intérêt, d’un groupement photoactivable permettant de créer, sous irradiation lumineuse, une liaison covalente avec la ou les protéine(s) cible(s) et d’une molécule de Biotine ou de Desthiobiotine afin d’extraire les protéines marquées des lysats cellulaires. Une fois marquées et capturées sur des billes d’agarose Streptavidine, les protéines d’intérêt ont été séparées sur des gels SDS-PAGE colorés au nitrate d’argent ou au bleu de Coomassie. Les bandes correspondantes ont été excisées, digérées à la trypsine et les peptides obtenus analysés par spectrométrie de masse. L’interrogation de la base de données Swissprot avec les données issues des expériences menées avec la sonde ITB5-P2 a permis d’identifier 3 protéines interagissant visiblement avec ce composé. Les expériences basées sur le composé ITB2 ont du être suspendues par manque de temps mais des résultats encourageants ont déjà été obtenus. Une bande pouvant correspondre à une protéine marquée spécifiquement par la sonde ITB2-P1 a en effet pu être observée en Western-blot suite à une première expérience de capture sur billes. Elle n’a toutefois pas pu être visualisée sur gel du fait d’une présence trop importante de protéines captées non spécifiquement par les billes. Les conditions expérimentales de capture ayant été optimisées avec le composé ITB5, leur application au composé ITB2 devrait maintenant permettre d’obtenir des gels plus propres à partir desquels la bande d’intérêt pourra être excisée pour être, elle aussi, analysée par spectrométrie de masse
An important breakthrough in the understanding of the mechanisms governing the process of iodide transport inside thyroid cells has been the cloning in 1996 of the protein responsible for this transport : the Na/I symporter (NIS). Different studies have been conducted ever since in order characterize this protein as well as the mechanisms which regulate its expression and its activity. Nevertheless, the cellular mechanisms of transport regulation and the proteins implied in the posttranslational regulation of the symporter remain largely unknown. The full understanding of these mechanisms would allow the treatment improvement of a lot of patients. Iodide transport is indeed involved not only in different thyroid pathologies, but also in radioactive iodide contaminations following nuclear accidents and in promising anticancer strategies by gene transfer. Chemogenomics, also called chemical genetics, is a multidisciplinary approach which goal is to explore the living systems thanks to small organic molecules. To better understand the mechanisms which govern iodide transport, our laboratory has set up a direct chemical genetic strategy which allowed us first to discover 10 molecules able to inhibit iodide transport. The objective of this thesis was to identify the protein targets of two molecules : ITB5 and ITB2. Electrophysiological and isotopic flux studies showed that these two molecules have a different mechanism of action. Their study should then allow the identification of at least two proteins involved in iodide transport.To identify the protein targets of ITB5 and ITB2, different probes were synthesized. These probes are made from the compound of interest, a photoactivable group allowing the creation, under light irradiation, of a covalent bound with the protein target(s) and a Biotin or Desthiobiotine molecule to extract the labeled proteins from cellular lysates. Once labeled and captured on agarose-Streptavidin beads, the proteins of interest were separated on SDS-PAGE gels stained either with silver nitrate or Coomassie blue. The corresponding bands were excised, digested by trypsin and the obtained peptides analyzed by mass spectrometry. A query made in the data bank Swissprot with the data obtained after the experiments conducted with the probe ITB5-P2 allowed us to identify 3 proteins apparently interacting with the compound ITB5. The experiments based on ITB2 had to be suspended because of a lack of time but encouraging results have been obtained. A band which may correspond to a protein specifically labeled by the probe ITB2-P1 has indeed been observed on a Western-blot after a first on-bead capture experiment. However, we couldn’t visualize it on a gel because of the important presence of proteins captured non specifically by the beads. The capture experimental conditions were optimized with the compound ITB5. These conditions will now be applied to the compound ITB2 and this should allow us to obtain cleaner gels on which the band of interest will be excised for an analyze by mass spectrometry

El objetivo principal de este trabajo es desarrollar un modelo sencillo que permita la caracterización hidrodinámica de membranas selectivas integradas en sistemas bi-iónicos, mediante la determinación de coeficientes de difusión y de espesores de las capas límite alrededor de la membrana. A tal fin, se empleó una célula de difusión rotatoria (CDR), que permite el establecimiento de condiciones hidrodinámicas bien definidas para el sistema de membrana, dado que la variación de la frecuencia de giro del cilindro interior (ω), permite disminuir el espesor de la capa límite sobre la membrana, lo que favorece el intercambio iónico a su través. Se puede comprobar éste comportamiento, mediante consideraciones en torno al coeficiente de difusión de los cationes en el sistema de membrana y del cálculo del propio espesor de la capa límite. El mencionado coeficiente se obtendrá a partir del flujo iónico en la membrana, determinado a partir de medidas de pH, junto a medidas de conductividad, en la fase externa (receptora), a diferentes temperaturas y a distintas valores de ω.La medida de los flujos, una vez establecida su dependencia con ω, permite obtener los coeficientes de difusión catiónicos en el sistema de membrana, en función de la temperatura y de ω. Las medidas de la conductividad permiten testar el modelo propuesto, mediante su correlación con los valores de pH obtenidos, proporcionando información adicional acerca de los coeficientes de difusión de los cationes.
From the experimental study of the ionic transport through selective membranes in biionic systems, a simple model which allows the characterising hydrodynamic of the membrane systems through the determination of diffusion coefficients and the thickness of the limit layer has been developed. With this purpose, a rotating diffusion cell that allows the setting of hydrodynamic conditions clearly for the membrane system has been used, studying the variation of the conductivity and the pH in the external phase (receiving) at different temperatures from 20ºC to 50ºC and at different rotating velocities ω. The measurement of the fluxes, once set its dependence with ω, allows obtained the diffusion coefficients cationics in the membrane system in accordance with the temperature and ω. The measurements of the conductivity allow the testing of this model, through its correlation with the values of the pH measured, obtaining additional data about the diffusion coefficient of the cations in the receiving phase.

ECF transporters are a group of newly defined ABC-like modular transporters and they are composed of three main elements: 1) a high-affinity membrane-embedded substrate binding protein (S component), 2) a membrane-spanning protein (T component), and 3) two identical or homologous ATPases (A, A’components) which resemble the nucleotide binding domains in ABC transporters. Staphylococcus aureus biotin transporter (SaBioMNY) belongs to the subgroup II ECF transporters which are characterized by their shared use of energy coupling module (AT module) by several S components, with each having a different substrate preference. Therefore, characterizing the S families in ECF transporters are important for us to gain new knowledge about the mechanism of subgroup II ECF transporters. Besides, laboratory has developed a series of biotin analogues with antibacterial activity against S. aureus. Previous studies have demonstrated that these compounds were capable of binding to the S component of S. aureus(i.e.SaBioY). It was reasonable to speculate that these biotin analogues were transported across the S. aureus cells by the biotin transporter BioY. To further improve the antibacterial potency and selectivity, the binding and translocation mode of these compounds across the bacterial membrane via SaBioY needs to be defined. By utilizing a filter disk diffusion assay, I determined that the susceptibility of E. coli BL21 to antibiotics (erythromycin, streptomycin and chloramphenicol) was significantly increased when wild type SaBioY was heterologously overexpressed in the cells. A library of SaBioY mutants was also screened in this assay and the overexpression of all the mutants surprisingly increased the sensitivity of E.coli cells to all three antibiotics compared to the un-induced one. One exceptional mutant was the D157K/K160E that was able to restore the tolerance of cells to the antimicrobial agents. I reasoned recombinant SaBioY adopted a functional channel in the membrane of E. coli for low molecular weight antibiotics to diffuse through. In addition, I also found that R75, D157 and K160 are essential to the surrogate transport pathway since a single amino acid change can dramatically alter the sensitivity of E. coli cells to antibiotics compared to the wild type one. To further characterize the biotin core transporter SaBioY, I attempted to purify recombinant SaBioY from E. coliBL21 (DE3). The optimized conditions for expressing SaBioY were determined to be 1) culturing cells at 25°C, 2) using the richer potassium buffered TB growth medium and 3) using a high concentration of IPTG (0.8 mM). I have also developed a system for the scalable purification of this integral membrane protein using SDS as a solubilizer. 9.7mg of SDS-solubilized SaBioY (with expected molecular weight of 19,492 Da) was obtained from 2 liters of culture after IMAC purification, with 90% purity determined by Commassie staining gel. A small panel of available mild detergents was subsequently tested for their efficiency of extracting membrane protein from natural lipids with TrionX-100 giving the best extraction efficiency. This present study paves the way for further detergent screening and purification of SaBioY.
Thesis (M.Phil.) -- University of Adelaide, School of Molecular and Biomedical Science, 2014

The clinically relevant human pathogen Staphylococcus aureus employs an energy coupling factor (ECF) transporter to import the important micronutrient biotin. Like the well characterised ABC transporters, the ECF transporters utilise the hydrolysis of ATP to move substrates across biological membranes. However, the ECF transporters do not use a solute-binding protein to bind substrate instead employing a membrane embedded protein to fulfil this role. In certain bacteria, the substrate binding protein required for binding biotin is known as BioY. The aim of this thesis was to investigate protein structure and function relationships involving the BioY protein from S. aureus (SaBioY). S. aureus has functional import and export processes that result in a biphasic profile of biotin uptake. Active translocation of biotin was temperature-dependent, optimum at 30 minutes, and inhibited by biotin or structural analogues of the vitamin. The study demonstrated that recombinant SaBioY could be expressed in E. coli, and was localised to the membrane fraction as observed by Western blot analysis on fractionated cell lysates and fluorescence microscopy. Importantly, a convenient ligand binding assay was developed that facilitated deeper analysis of the SaBioY structure and function. SaBioY primarily recognises the ureido ring of biotin for substrate capture, but an intact thiophene ring also aids binding. Although a variety of functional groups can be appended onto the carboxyl group of the biotin moiety, the linker used to connect the molecules and the chemical property of functional group can impact binding to SaBioY. This knowledge can be exploited for developing biotin-based analogues with applications in antibiotic drug discovery. Since an X-crystal structure of SaBioY is not available, membrane topology predictions and computational modelling were used to generate a molecular module of SaBioY. This yielded a model containing 5 transmembrane domains, 3 extracellular loops, and 1 intracellular loop with intracellular N- and C-termini. Whilst the model was in good agreement with known crystal structures of other known S components, it possessed an additional V-shaped membrane embedded helix. Conserved amino acid residues in BioY were identified using the web-based Clustal-W alignment program and then mapped onto the SaBioY model. A series of 24 SaBioY mutants were then generated using random and site-directed mutagenesis approaches. Fluorescence polarisation based competitive-binding assays using a fluorescent-biotin tracer revealed several conserved (R75, D157 and K160) and non-conserved (N38, T54, F81, F88 and D128) residues important for biotin binding. Interestingly, a double mutant D157K/K160E completely abolished biotin binding. A filter disk diffusion assay using a panel of antibiotics showed recombinant expression of SaBioY increased E. coli antibiotic sensitivity to streptomycin, erythromycin and chloramphenicol, probably by forming a pore through channel by dimerisation requiring a dynamic cooperative interaction. Expression of all of the SaBioY mutants increased sensitivity to the three antibiotics. The D157K/K160E double mutant was an exception, as it had no effect upon antibiotic sensitivity. We proposed that D157 and K160 together play an essential role in SaBioY activity. In conclusion, this study successfully characterised the SaBioY transporter in both its native state and using a recombinant E. coli expression system. Substrate specificity of the transporter was determined, as was the channel gating potency of SaBioY for certain antibiotics when expressed in E. coli. Computational modelling and a novel FP based competitive assay also provided useful tools for biochemical analysis of SaBioY structure and function relationships. Further studies are now required to determine the SaBioY X-ray crystal structure, transport mechanism and regulation as well as to explore possible application of the transporter as a novel drug target or an alternative gating system new antibiotic agents.
Thesis (Ph.D.) -- University of Adelaide, School of Biological Sciences, 2015.

Tese de Doutoramento em Ciências (Especialidade em Biologia)
Os açúcares desempenham funções vitais nos seres vivos, principalmente como fontes de carbono e de energia, mas também como reguladores osmóticos e como moléculas sinalizadoras. Em particular, na videira (Vitis vinífera L.), a qualidade do vinho depende dos níveis de açúcar nos bagos de uva porque determinam a concentração em etanol e influenciam a síntese de compostos secundários (incluindo pigmentos). Diferentes famílias de transportadores membranares presentes no genoma das plantas desempenham um papel essencial na translocação de açúcares entre os tecidos fotossintéticos e os tecidos de armazenamento. Entre eles, os transportadores denominados SWEET (Sugars Will Eventually be Exported Transporter), recentemente identificados, têm revelado diferentes papéis em mecanismos fisiológicos onde o efluxo de açúcar é fundamental, como nos nectários. Na videira, a família SWEET compreende 17 membros. No presente estudo pretendeu-se elucidar o papel dos VvSWEETs na resposta da videira à infeção por fungos (Botrytis cinerea e Erysiphe necator) e ao stresse abiótico, incluindo a secura. Além disso, para estudar os papéis fisiológicos do VvSWEET7 e VvSWEET15, foram aplicadas diferentes técnicas de engenharia genética de plantas, tal como CRISPR-Cas9. Em particular, na variedade Trincadeira, susceptível ao fungo B. cinerea, e na variedade Carignan, susceptível a E. necator, foram analisadas em detalhe as modificações no perfil de expressão dos SWEETs em bagos de uva infetados. Os resultados mostraram que a infeção por E. necator causa modificações mais pronunciadas na expressão dos VvSWEETs do que a infecção por Botrytis. Por outro lado, a maioria dos SWEETs da videira foram regulados negativamente em bagos de uva em resposta à secura, no entanto, o VvSWEET10 e VvSWEET11 foram regulados positivamente. Foi também observado que a expressão do VvSWEET1, VvSWEET4 e VvSWEET11 é regulada positivamente em folhas tratadas com caulino (filme inerte usado para proteger as videiras em situações de deficit hídrico, de radiação solar extrema e de ondas de calor), sugerindo que este mineral estimula a capacidade de transporte de sacarose entre os tecidos fotossintéticos e os tecidos de armazenamento. Estudos subsequentes mostraram que os genes VvSWEET11 e VvSWEET15 são positivamente regulados em bagos de uva submetidos a temperaturas de 50ºC durante 7 dias, tratamento normalmente usado para a produção de uvas passas. Uma vez que os níveis de transcritos dos genes VvSWEET7 e VvSWEET15 foram elevados nos bagos de uva e aumentaram em resposta à infeção por Botrytis, as proteínas VvSWEET7 e VvSWEET15 foram alvo de estudos adicionais para se avaliar a sua localização sub-celular e função. As proteínas de fusão VvSWEET7-GFP e VvSWEET15- GFP foram transitoriamente expressas em células da epiderme de Nicotiana benthamiana e os resultados de microscopia confocal mostraram que ambas as proteínas se localizam claramente na membrana plasmática. Após expressão heteróloga numa estirpe mutante de Saccharomyces cerevisiae (hxt-null), a proteína VvSWEET7 foi caracterizada funcionalmente como um transportador de glucose e de sacarose (Km =15,4 mM glucose e Km = 40,1 mM sacarose). Ensaios de inibição competitiva mostraram que o manitol e o sorbitol inibem o transporte de D-[14C(U)]- glucose, sugerindo que, além de mono- e de dissacarídeos, o VvSWEET7 medeia o transporte de polióis. No presente trabalho foram ainda identificados no genoma da videira 18 membros da família de transportadores de açúcares denominada ERD6like e a proteína VvERD6l13 foi alvo de um estudo mais aprofundado. A proteína de fusão VvERD6l13-GFP foi transitoriamente expressa em folhas de N. benthamiana após transformação mediada por Agrobacterium e os resultados de microscopia de fluorescência mostraram que se localiza na membrana plasmática. Estudos de transporte de açúcares marcados radioativamente, após expressão heteróloga em leveduras mutantes (hxt-null), mostraram que a proteína VvERD6l13 é um transportador de sacarose com protões (Km = 33 mM). O gene VvERD6l13 é fortemente regulado em bagos de uva infetados com Botrytis ou E. necator, sugerindo que a proteína VvERD6l13 tem um papel importante durante a interação planta-patógeno. Além disso, o VvERD6l13 é expresso em diferentes tecidos da videira, em particular na raiz. Genericamente, os resultados mostraram que os transportadores VvSWEET e o VvERD6l desempenham um papel importante na mobilização de açúcares durante o desenvolvimento dos bagos de uva e que a sua expressão é regulada ao nível da transcrição em resposta ao stresse biótico e abiótico. No seu conjunto, estes resultados ajudam a compor o puzzle complexo dos mecanismos de resposta da videira aos stresses biótico e abiótico, abrindo ainda caminhos novos e desafiadores no tópico do transporte transmembranar em plantas.
Sugars perform vital functions in the living world, primarily as sources of carbon and energy, but also as osmotic regulators and signaling molecules, among others. This is particularly relevant in the grapevine (Vitis vinifera L.) as the quality of the wine depends on the sugar concentration in the grape berry as it determines the final concentration in ethanol, but is also tightly related to the amount of secondary compounds (including pigments) synthesized during ripening. Different sugar transporter families are present in the genome of plants to fulfill the task of transmembrane sugar transport, which is pivotal for long distance transport between sources and sinks. Among these, the newly identified SWEETs transporters (from Sugars Will Eventually be Exported Transporter) have important roles in numerous physiological mechanisms where sugar efflux is critical. In grapevine, the SWEET family comprises 17 members. In this study, the main objective was to elucidate the role of VvSWEETs in grapevine response to fungal attack (Botrytis cinerea or Erysiphe necator infection) and abiotic stress, including drought. Also, to further study the physiological roles of VvSWEET7 and VvSWEET15, different plant genetic engineering techniques, such as CRISPRCas9, were used. In the B. cinerea-susceptible cv. Trincadeira and in the E. necator-susceptible cv. Carignan, modifications in the gene expression profile of SWEETs in infected grape berries were thoroughly analyzed. Overall, results showed that E. necator infection caused more pronounced modifications in VvSWEET gene expression than Botrytis infection. Moreover, the majority of grapevine SWEET genes were down-regulated in berries from droughtstressed vines of cv. Tempranillo, while VvSWEET10 and VvSWEET11 were up-regulated. In kaolin-treated leaves the expression of VvSWEET1, VvSWEET4 and VvSWEET11 was up-regulated, suggesting that this chemically inert mineral used to protect vines from radiation, drought and heat stimulates sucrose transport capacity improving source-to-sink transport of sucrose. Results also showed that VvSWEET11 and VvSWEET15 were strongly up-regulated in berries subjected to 50ºC during 7 days, a protocol normally used to produce raisins. Following the observation that VvSWEET7 and VvSWEET15 were strongly expressed in berries and clearly up-regulated in response to Botrytis infection in cv. Trincadeira, they were subjected to additional studies to evaluate the subcellular localization and function of the encoded proteins. VvSWEET7-GFP and VvSWEET15-GFP fusion proteins were transiently expressed in Nicotiana benthamiana epidermal cells after Agrobacterium-mediated transformation and both proteins clearly localized to the plasma membrane, as assessed by confocal microscopy. VvSWEET7 was functionally characterized after overexpression in an hxt-null Saccharomyces cerevisiae strain as a low-affinity, high-capacity glucose and sucrose transporter, with a Km of 15.4 mM for glucose and 40.1 mM for sucrose. Competitive inhibition experiments showed that mannitol and sorbitol also inhibited D-[14C(U)]-glucose transport, suggesting that, besides mono- and disaccharides, VvSWEET7 mediates the transport of polyols. In the grapevine genome 18 members of the sugar transporter family ERD6l were identified and VvERD6l13 was selected for further characterization. The fusion protein VvERD6l13-GFP was transiently expressed in N. benthamiana leaves after Agrobacterium-mediated transformation. VvERD6l13 is localized in the plasma membrane. When VvERD6l13 was heterologously expressed in an hxt-null S. cerevisiae strain, it was observed that the protein mediates H+-dependent sucrose transport with a Km = 33 mM. VvERD6l13 is strongly up-regulated in infected grape berries with Botrytis or E. necator, suggesting that it plays an important role during pathogen-host plant interaction. Moreover, VvERD6l13 is expressed in different grapevine tissues, but its steady-state transcript levels were particularly high in roots. In sum, VvSWEET and VvERD6l transporters are important players in sugar mobilization during grape berry development and their expression is transcriptionally reprogrammed in response to biotic and abiotic stress. Together, these results constitute a new piece of the complex puzzle that is grapevine interaction with its surrounding environment and existing biological threats, while also opening new and exciting pathways in the plant sugar transporter research topic.
Fundação para a Ciência e Tecnologia (FCT) - Bolsa de Doutoramento (PD/BD/113616/2015), integrada no Programa Doutoral “Agricultural Production Chains-From Fork to Farm (AgriChains) (PD/00122/2012). Fundação para a Ciência e Tecnologia (FCT) e Fundos Europeus (FEDER/POCI /COMPETE2020) – Projecto “MitiVineDrought - Combining "omics" with molecular, biochemical and physiological analyses as an integrated effort to validate novel and easy-to-implement drought mitigation strategies in grapevine while reducing water use” (PTDC/BIA-FBT/30341/2017 and POCI-01-0145-FEDER-030341). Fundação para a Ciência e Tecnologia (FCT) e Fundos Europeus (FEDER/POCI /COMPETE2020) – Projecto “BerryPlastid - Biosynthesis of secondary compounds in the grape berry: unlocking the role of the plastid” (POCI-01-0145-FEDER-028165 and PTDC/BIA-FBT/28165/2017). Fundação para a Ciência e Tecnologia (FCT) – Projecto “GrapInfectomics - Transcriptome and metabolome reprogramming in Vitis vinifera cv. Aragonês and Vitis rupestris berries upon infection with Erysiphe necator” (PTDC/ASP-HOR/28485/2017). Fundação para a Ciência e Tecnologia (FCT) – Projecto “CherryCrackLess” - Cherry cracking & mitigation strategies: towards their understanding using a functional metabolomic approach” (PTDC/AGRPRO/ 7028/2014).