Analysis of VirB4, a membrane-associated ATPase subunit essential for T -complex transport in Agrobacterium tumefaciens

Agrobacterium tumefaciens is a plant pathogen with the unique ability to export oncogenic DNA-protein complexes (T-complexes) to susceptible plant cells and cause crown gall tumors. Delivery of the T-complexes across the bacterial membranes requires eleven VirB proteins and VirD4, which are postulated to form a transmembrane transporter. This thesis examines the subcellular localization and oligomeric structure of the 87-kDa VirB4 protein, which is one of three essential ATPases proposed to energize T-complex transport and/or assembly. Results of subcellular localization studies showed that VirB4 is tightly associated with the cytoplasmic membrane, suggesting that it is a membrane-spanning protein. The membrane topology of VirB4 was determined by using a nested deletion strategy to generate random fusions between virB4 and the periplasmically-active alkaline phosphatase, $\sp\prime phoA$. Analysis of PhoA and complementary $\beta$-galactosidase reporter fusions identified two putative periplasmically-exposed regions in VirB4. A periplasmic exposure of one of these regions was further confirmed by protease susceptibility assays using A. tumefaciens spheroplasts. To gain insight into the structure of the transporter, the topological configurations of other VirB proteins were also examined. Results from hydropathy analyses, subcellular localization, protease susceptibility, and PhoA reporter fusion studies support a model that all of the VirB proteins localize at one or both of the bacterial membranes. Immunoprecipitation and Co$\sp{2+}$ affinity chromatography studies demonstrated that native VirB4 (87-kDa) and a functional N-terminally tagged HIS-VirB4 derivative (89-kDa) interact and that the interaction is independent of other VirB proteins. A $\lambda$ cI repressor fusion assay supplied further evidence for VirB4 dimer formation. A VirB4 dimerization domain was localized to the N-terminal third of the protein, as judged by: (i) transdominance of an allele that codes for this region of VirB4; (ii) co-retention of a His-tagged N-terminal truncation derivative and native VirB4 on Co$\sp{2+}$ affinity columns; and (iii) dimer formation of the N-terminal third of VirB4 fused to the cI repressor protein. Taken together, these findings are consistent with a model that VirB4 is topologically configured as an integral cytoplasmic membrane protein with two periplasmic domains and that VirB4 assembles as homodimers via an N-terminal dimerization domain. Dimer formation is postulated to be essential for stabilization of VirB4 monomers during T-complex transporter assembly. ^

(Table 2) Sorting and mineral composition of sediment obtained near Sylt, North Sea

Carefully selected sea bottom surface fine sand samples were studied from two sand ribbons normal to the shore. Possible sediment transport along these sand ribbons were investigated from interpretation of the sediment patterns. Simple grain size parameters were obtained and results of heavy mineral and feldspar analysis were compared. On one ribbon offshore sediment movement was indicated, while conversely on the other, onshore movement is proposed.

¿Impact from road transport of radioactive wastes in Spain?

Transports of radioactive wastes in Spain are becoming issues of renewed interest, due to the increased mobility of these materials which can be expected after the building and operation of the planned central repository for this country in a near future. Such types of residues will be mainly of the medium and high activity classes and have raised concerns on the safety of the operations, the radiological protection of the individuals, the compliance with the legal regulations and their environmental consequences of all kind. In this study, relevant information for the assessment of radiological risk of road transport were taken into account, as the sources and destination of the radioactive transports, the amount of traveling to be done, the preferred routes and populations affected, the characterization of the residues and containers, their corresponding testing, etc. These data were supplied by different organizations fully related with these activities, like the nuclear power stations, the companies in charge of radioactive transports, the enterprises for inspection and control of the activities, etc., as well as the government institutions which are responsible for the selection and location of the storage facility and other decisions on the nuclear policies of the country. Thus, we have developed a program for computing the data in such a form that by entering the radiation levels at one meter of the transport loads and by choosing a particular displacement, the computer application is capable to calculate the corresponding radiological effects, like the global estimated impact, its relevance to the population in general or on those people living and driving near the main road routes, the doses received by the most exposed individuals (e.g. the workers for loading or driving the vehicle), or the probability of detrimental on the human health. The results of this work could be of help for a better understanding and management of these activities and their related impacts; at the same time that the generated reports of the computer application are considered of particular interest as innovative and complementary information to the current legal documentation, which is basically required for transporting radioactive wastes in the country, according with the international safety rules (like IAEA and ADR).Though main studies are still in progress, as the definite location for the Spanish storage facility has not been decided yet, preliminary results with the existing transports of residues of medium activity indicate that the radiological impact is very low in conventional operations. Nevertheless, the management of these transports is complex and laborious, making it convenient to progress further in the analysis and quantification of this kind of events, which constitutes one of the main objectives of the present study for the radioactive road mobility in Spain.

Optimal and Long-Term Dynamic Transport Policy Design: Seeking Maximum Social Welfare through a Pricing Scheme.

This article presents an alternative approach to the decision-making process in transport strategy design. The study explores the possibility of integrating forecasting, assessment and optimization procedures in support of a decision-making process designed to reach the best achievable scenario through mobility policies. Long-term evaluation, as required by a dynamic system such as a city, is provided by a strategic Land-Use and Transport Interaction (LUTI) model. The social welfare achieved by implementing mobility LUTI model policies is measured through a cost-benefit analysis and maximized through an optimization process throughout the evaluation period. The method is tested by optimizing a pricing policy scheme in Madrid on a cordon toll in a context requiring system efficiency, social equity and environmental quality. The optimized scheme yields an appreciable increase in social surplus through a relatively low rate compared to other similar pricing toll schemes. The results highlight the different considerations regarding mobility impacts on the case study area, as well as the major contributors to social welfare surplus. This leads the authors to reconsider the cost-analysis approach, as defined in the study, as the best option for formulating sustainability measures.

Models for Internal Quality Fruit Sorting, Based on Time- Domain Laser Reflectance Spectroscopy (TDRS)

Time domain laser reflectance spectroscopy (TDRS) was applied for the first time to evaluate internal fruit quality. This technique, known in medicine-related knowledge areas, has not been used before in agricultural or food research. It allows the simultaneous non-destructive measuring of two optical characteristics of the tissues: light scattering and absorption. Models to measure firmness, sugar & acid contents in kiwifruit, tomato, apple, peach, nectarine and other fruits were built using sequential statistical techniques: principal component analysis, multiple stepwise linear regression, clustering and discriminant analysis. Consistent correlations were established between the two parameters measured with TDRS, i.e. absorption & transport scattering coefficients, with chemical constituents (sugars and acids) and firmness, respectively. Classification models were built to sort fruits into three quality grades, according to their firmness, soluble solids and acidity.

Mesh traversal and sorting for efficient memory usage in scientific codes

Applications that operate on meshes are very popular in High Performance Computing (HPC) environments. In the past, many techniques have been developed in order to optimize the memory accesses for these datasets. Different loop transformations and domain decompositions are com- monly used for structured meshes. However, unstructured grids are more challenging. The memory accesses, based on the mesh connectivity, do not map well to the usual lin- ear memory model. This work presents a method to improve the memory performance which is suitable for HPC codes that operate on meshes. We develop a method to adjust the sequence in which the data are used inside the algorithm, by means of traversing and sorting the mesh. This sorted mesh can be transferred sequentially to the lower memory levels and allows for minimum data transfer requirements. The method also reduces the lower memory requirements dra- matically: up to 63% of the L1 cache misses are removed in a traditional cache system. We have obtained speedups of up to 2.58 on memory operations as measured in a general- purpose CPU. An improvement is also observed with se- quential access memories, where we have observed reduc- tions of up to 99% in the required low-level memory size.

Robustness and Recoverability in Transport Logistics

Abstract Transport is the foundation of any economy: it boosts economic growth, creates wealth, enhances trade, geographical accessibility and the mobility of people. Transport is also a key ingredient for a high quality of life, making places accessible and bringing people together. The future prosperity of our world will depend on the ability of all of its regions to remain fully and competitively integrated in the world economy. Efficient transport is vital in making this happen. Operations research can help in efficiently planning the design and operating transport systems. Planning and operational processes are fields that are rich in combinatorial optimization problems. These problems can be analyzed and solved through the application of mathematical models and optimization techniques, which may lead to an improvement in the performance of the transport system, as well as to a reduction in the time required for solving these problems. The latter aspect is important, because it increases the flexibility of the system: the system can adapt in a faster way to changes in the environment (i.e.: weather conditions, crew illness, failures, etc.). These disturbing changes (called disruptions) often enforce the schedule to be adapted. The direct consequences are delays and cancellations, implying many schedule adjustments and huge costs. Consequently, robust schedules and recovery plans must be developed in order to fight against disruptions. This dissertation makes contributions to two different fields: rail and air applications. Robust planning and recovery methods are presented. In the field of railway transport we develop several mathematical models which answer to RENFE’s (the major railway operator in Spain) needs: 1. We study the rolling stock assignment problem: here, we introduce some robust aspects in order to ameliorate some operations which are likely to fail. Once the rolling stock assignment is known, we propose a robust routing model which aims at identifying the train units’ sequences while minimizing the expected delays and human resources needed to perform the sequences. 2. It is widely accepted that the sequential solving approach produces solutions that are not global optima. Therefore, we develop an integrated and robust model to determine the train schedule and rolling stock assignment. We also propose an integrated model to study the rolling stock circulations. Circulations are determined by the rolling stock assignment and routing of the train units. 3. Although our aim is to develop robust plans, disruptions will be likely to occur and recovery methods will be needed. Therefore, we propose a recovery method which aims to recover the train schedule and rolling stock assignment in an integrated fashion all while considering the passenger demand. In the field of air transport we develop several mathematical models which answer to IBERIA’s (the major airline in Spain) needs: 1. We look at the airline-scheduling problem and develop an integrated approach that optimizes schedule design, fleet assignment and passenger use so as to reduce costs and create fewer incompatibilities between decisions. Robust itineraries are created to ameliorate misconnected passengers. 2. Air transport operators are continuously facing competition from other air operators and different modes of transport (e.g., High Speed Rail). Consequently, airline profitability is critically influenced by the airline’s ability to estimate passenger demands and construct profitable flight schedules. We consider multi-modal competition including airline and rail, and develop a new approach that estimates the demand associated with a given schedule; and generates airline schedules and fleet assignments using an integrated schedule design and fleet assignment optimization model that captures the impacts of schedule decisions on passenger demand.

System supporting public transport based on geolocation

El propósito de este proyecto de fin de Grado es el estudio y desarrollo de una aplicación basada en Android que proporcionará soporte y atención a los servicios de transporte público existentes en Cracovia, Polonia. La principal funcionalidad del sistema será consultar la posición de un determinado autobús o tranvía y mostrar su ubicación con exactitud. Para lograr esto, necesitaremos tres fases de desarrollo. En primer lugar, deberemos implementar un sistema que obtenga las coordenadas geográficas de los vehículos de transporte público en cada instante. A continuación, tendremos que registrar todos estos datos y almacenarlos en una base de datos en un servidor web. Por último, desarrollaremos un sistema cliente que realice consultas a tiempo real sobre estos datos almacenados, obteniendo la posición para una línea determinada y mostrando su ubicación con un marcador en el mapa. Para hacer el seguimiento de los vehículos, sería necesario tener acceso a una API pública que nos proporcionase la posición registrada por los GPS que integran cada uno de ellos. Como esta API no existe actualmente para los servicios de autobús, y para los tranvías es de uso meramente privado, desarrollaremos una segunda aplicación en Android que hará las funciones del lado servidor. En ella podremos elegir mediante una simple interfaz el número de línea y un código específico que identificará a cada vehículo en particular (e.g. podemos tener 6 tranvías recorriendo la red al mismo tiempo para la línea 24). Esta aplicación obtendrá las coordenadas geográficas del teléfono móvil, lo cual incluye latitud, longitud y orientación a través del proveedor GPS. De este modo, podremos realizar una simulación de como el sistema funcionará a tiempo real utilizando la aplicación servidora desde dentro de un tranvía o autobús y, al mismo tiempo, utilizando la aplicación cliente haciendo peticiones para mostrar la información de dicho tranvía. El cliente, además, podrá consultar la ruta de cualquier línea sin necesidad de tener acceso a Internet. Almacenaremos las rutas y paradas de cada línea en la memoria del teléfono móvil utilizando ficheros XML debido al poco espacio que ocupan y a lo útil que resulta poder consultar un trayecto en cualquier momento, independientemente del acceso a la red. El usuario también podrá consultar las tablas de horarios oficiales para cada línea. Aunque en este caso si será necesaria una conexión a Internet debido a que se realizará a través de la web oficial de MPK. Para almacenar todas las coordenadas de cada vehículo en cada instante necesitaremos crear una base de datos en un servidor. Esto se resolverá mediante el uso de MYSQL y PHP. Se enviarán peticiones de tipo GET y POST a los servicios PHP que se encargarán de traducir y realizar la consulta correspondiente a la base de datos MYSQL. Por último, gracias a todos los datos recogidos relativos a la posición de los vehículos de transporte público, podremos realizar algunas tareas de análisis. Comparando la hora exacta a la que los vehículos pasaron por cada parada y la hora a la que deberían haber pasado según los horarios oficiales, podremos descubrir fallos en estos. Seremos capaces de determinar si es un error puntual debido a factores externos (atascos, averías,…) o si por el contrario, es algo que ocurre muy a menudo y se debería corregir el horario oficial. ABSTRACT The aim of this final Project (for University) is to develop an Android application thatwill provide support and feedback to the public transport services in Krakow. The main functionality of the system will be to track the position of a desired bus or tram line, and display its position on the map. To achieve this, we will need 3 stages: the first one will be to implement a system that sends the geographical position of the public transport vehicles, the second one will be to collect this data in a web server, and the last one will be to get the last location registered for the desired line and display it on the map. For tracking the vehicles, we would need to have access to a public API that should be connected with each bus/tram GPS. As this doesn’t exist in Krakow or at least is not available for public use, we will develop a second android application that will do the server side job. We will be able to choose in a simple interface the line number and a code letter to identify each vehicle (e.g. we can have 6 trams that belong to the line number 24 working at the same time). It will take the current mobile geolocation; this includes getting latitude, longitude and bearing from the GPS provider. Thus, we will be able to make a simulation of how the system works in real time by using the server app inside a tram and at the same time, using the client app and making requests to display the information of that tram. The client will also be able to check the path of the desired line without internet access. We will store the path and stops for each line locally in the phone memory using xml files due to the few requirements of available space it needs and the usefulness of checking a path when needed. This app will also offer the functionality of checking the timetable for the line, but in this case, it will link to the official Mpk website, so Internet access will be required. For storing all the coordinates for each vehicle at every moment we will need to create a database on a server. We have decided that the easiest way is to use Mysql and PHP for the deployment of the service. We will send GET and POST requests to the php files and those files will make the according queries to our database. Finally, based on all the collected data, we will be able to get some information about errors in the system of public transport timetables. We will check at what time a line was in each specific stop and compare it with the official timetable to find mistakes of time. We will determine if it is something that happens occasionally and related to external factors (e.g. traffic jams, breakdowns…) or if on the other hand, it is something that happens very often and the public transport timetables should be looked over and corrected.

Optimal and Long-Term Dynamic Transport Policy Design: Seeking Maximum Social Welfare through a Pricing Scheme

This article presents an alternative approach to the decision-making process in transport strategy design. The study explores the possibility of integrating forecasting, assessment and optimization procedures in support of a decision-making process designed to reach the best achievable scenario through mobility policies. Long-term evaluation, as required by a dynamic system such as a city, is provided by a strategic Land-Use and Transport Interaction (LUTI) model. The social welfare achieved by implementing mobility LUTI model policies is measured through a cost-benefit analysis and maximized through an optimization process throughout the evaluation period. The method is tested by optimizing a pricing policy scheme in Madrid on a cordon toll in a context requiring system efficiency, social equity and environmental quality. The optimized scheme yields an appreciable increase in social surplus through a relatively low rate compared to other similar pricing toll schemes. The results highlight the different considerations regarding mobility impacts on the case study area, as well as the major contributors to social welfare surplus. This leads the authors to reconsider the cost-analysis approach, as defined in the study, as the best option for formulating sustainability measures.

TITIM GIS-tool: A GIS-based decision support system for measuring the territorial impact of transport infrastructures

To achieve sustainability in the area of transport we need to view the decision-making process as a whole and consider all the most important socio-economic and environmental aspects involved. Improvements in transport infrastructures have a positive impact on regional development and significant repercussions on the economy, as well as affecting a large number of ecological processes. This article presents a DSS to assess the territorial effects of new linear transport infrastructures based on the use of GIS. The TITIM ? Transport Infrastructure Territorial Impact Measurement ? GIS tool allows these effects to be calculated by evaluating the improvement in accessibility, loss of landscape connectivity, and the impact on other local territorial variables such as landscape quality, biodiversity and land-use quality. The TITIM GIS tool assesses these variables automatically, simply by entering the required inputs, and thus avoiding the manual reiteration and execution of these multiple processes. TITIM allows researchers to use their own GIS databases as inputs, in contrast with other tools that use official or predefined maps. The TITIM GIS-tool is tested by application to six HSR projects in the Spanish Strategic Transport and Infrastructure Plan 2005?2020 (PEIT). The tool creates all 65 possible combinations of these projects, which will be the real test scenarios. For each one, the tool calculates the accessibility improvement, the landscape connectivity loss, and the impact on the landscape, biodiversity and land-use quality. The results reveal which of the HSR projects causes the greatest benefit to the transport system, any potential synergies that exist, and help define a priority for implementing the infrastructures in the plan

Time-multiscale methods for the simulation of slow transport problems in atomistic systems

En esta tesis presentamos una teoría adaptada a la simulación de fenómenos lentos de transporte en sistemas atomísticos. En primer lugar, desarrollamos el marco teórico para modelizar colectividades estadísticas de equilibrio. A continuación, lo adaptamos para construir modelos de colectividades estadísticas fuera de equilibrio. Esta teoría reposa sobre los principios de la mecánica estadística, en particular el principio de máxima entropía de Jaynes, utilizado tanto para sistemas en equilibrio como fuera de equilibrio, y la teoría de las aproximaciones del campo medio. Expresamos matemáticamente el problema como un principio variacional en el que maximizamos una entropía libre, en lugar de una energía libre. La formulación propuesta permite definir equivalentes atomísticos de variables macroscópicas como la temperatura y la fracción molar. De esta forma podemos considerar campos macroscópicos no uniformes. Completamos el marco teórico con reglas de cuadratura de Monte Carlo, gracias a las cuales obtenemos modelos computables. A continuación, desarrollamos el conjunto completo de ecuaciones que gobiernan procesos de transporte. Deducimos la desigualdad de disipación entrópica a partir de fuerzas y flujos termodinámicos discretos. Esta desigualdad nos permite identificar la estructura que deben cumplir los potenciales cinéticos discretos. Dichos potenciales acoplan las tasas de variación en el tiempo de las variables microscópicas con las fuerzas correspondientes. Estos potenciales cinéticos deben ser completados con una relación fenomenológica, del tipo definido por la teoría de Onsanger. Por último, aportamos validaciones numéricas. Con ellas ilustramos la capacidad de la teoría presentada para simular propiedades de equilibrio y segregación superficial en aleaciones metálicas. Primero, simulamos propiedades termodinámicas de equilibrio en el sistema atomístico. A continuación evaluamos la habilidad del modelo para reproducir procesos de transporte en sistemas complejos que duran tiempos largos con respecto a los tiempos característicos a escala atómica. ABSTRACT In this work, we formulate a theory to address simulations of slow time transport effects in atomic systems. We first develop this theoretical framework in the context of equilibrium of atomic ensembles, based on statistical mechanics. We then adapt it to model ensembles away from equilibrium. The theory stands on Jaynes' maximum entropy principle, valid for the treatment of both, systems in equilibrium and away from equilibrium and on meanfield approximation theory. It is expressed in the entropy formulation as a variational principle. We interpret atomistic equivalents of macroscopic variables such as the temperature and the molar fractions, wich are not required to be uniform, but can vary from particle to particle. We complement this theory with Monte Carlo summation rules for further approximation. In addition, we provide a framework for studying transport processes with the full set of equations driving the evolution of the system. We first derive a dissipation inequality for the entropic production involving discrete thermodynamic forces and fluxes. This discrete dissipation inequality identifies the adequate structure for discrete kinetic potentials which couple the microscopic field rates to the corresponding driving forces. Those kinetic potentials must finally be expressed as a phenomenological rule of the Onsanger Type. We present several validation cases, illustrating equilibrium properties and surface segregation of metallic alloys. We first assess the ability of a simple meanfield model to reproduce thermodynamic equilibrium properties in systems with atomic resolution. Then, we evaluate the ability of the model to reproduce a long-term transport process in complex systems.

An integrated modeling approach to assess the impact on road freight transport demand of allowing longer and heavier vehicles (LHVs) in Spain

The assessment on introducing Longer and Heavier Vehicles (LHVs) on the road freight transport demand is performed in this paper by applying an integrated modeling approach composed of a Random Utility-Based Multiregional Input-Output model (RUBMRIO) and a road transport network model. The approach strongly supports the concept that changes in transport costs derived from the LHVs allowance as well as the economic structure of regions have both direct and indirect effects on the road freight transport system. In addition, we estimate the magnitude and extent of demand changes in the road freight transportation system by using the commodity-based structure of the approach to identify the effect on traffic flows and on pollutant emissions over the whole network of Spain by considering a sensitivity analysis of the main parameters which determine the share of Heavy-Goods Vehicles (HGVs) and LHVs. The results show that the introduction of LHVs will strengthen the competitiveness of the road haulage sector by reducing costs, emissions, and the total freight vehicles required.

The human homologue of Saccharomyces cerevisiae Gle1p is required for poly(A)+ RNA export

The mechanism of mRNA export is a complex issue central to cellular physiology. We characterized previously yeast Gle1p, a protein with a leucine-rich (LR) nuclear export sequence (NES) that is essential for poly(A)+ RNA export in Saccharomyces cerevisiae. To characterize elements of the vertebrate mRNA export pathway, we identified a human homologue of yeast Gle1p and analyzed its function in mammalian cells. hGLE1 encodes a predicted 75-kDa polypeptide with high sequence homology to yeast Gle1p, but hGle1p does not contain a sequence motif matching any of the previously characterized NESs. hGLE1 can complement a yeast gle1 temperature-sensitive export mutant only if a LR-NES is inserted into it. To determine whether hGle1p played a role in nuclear export, anti-hGle1p antibodies were microinjected into HeLa cells. In situ hybridization of injected cells showed that poly(A)+ RNA export was inhibited. In contrast, there was no effect on the nuclear import of a glucocorticoid receptor reporter. We conclude that hGle1p functions in poly(A)+ RNA export, and that human cells facilitate such export with a factor similar to yeast but without a recognizable LR-NES. With hGle1p localized at the nuclear pore complexes, hGle1p is positioned to act at a terminal step in the export of mature RNA messages to the cytoplasm.

Proteinase-activated receptor 2 (PAR2)-activating peptides: Identification of a receptor distinct from PAR2 that regulates intestinal transport

The effects of PAR2-activating PAR2-activating peptides, SLIGRL (SL)-NH2, and trans-cinnamoyl-LIGRLO (tc)-NH2 were compared with the action of trypsin, thrombin, and the PAR1 selective-activating peptide: Ala-parafluoroPhe-Arg-cyclohexylAla-Citrulline-Tyr (Cit)-NH2 for stimulating intestinal ion transport. These agonists were added to the serosa of stripped rat jejunum segments mounted in Ussing chambers, and short circuit current (Isc) was used to monitor active ion transport. The relative potencies of these agonists also were evaluated in two bioassays specific for the activation of rat PAR2: a cloned rat PAR2 cell calcium-signaling assay (PAR2-KNRK cells) and an aorta ring relaxation (AR) assay. In the Isc assay, all agonists, except thrombin, induced an Isc increase. The SL-NH2-induced Isc changes were blocked by indomethacin but not by tetrodotoxin. The relative potencies of the agonists in the Isc assay (trypsin≫SL-NH2>tc-NH2>Cit-NH2) were strikingly different from their relative potencies in the cloned PAR2-KNRK cell calcium assay (trypsin≫>tc-NH2 ≅ SL-NH2≫>Cit-NH2) and in the AR assay (trypsin≫>tc-NH2 ≅ SL-NH2). Furthermore, all agonists were maximally active in the PAR2-KNRK cell and AR assays at concentrations that were one (PAR2 -activating peptides) or two (trypsin) orders of magnitude lower than those required to activate intestinal transport. Based on the distinct potency profile for these agonists and the considerable differences in the concentration ranges required to induce an Isc effect in the intestinal assay compared with the PAR2-KNRK and AR assays, we conclude that a proteinase-activated receptor, pharmacologically distinct from PAR2 and PAR1, is present in rat jejunum and regulates intestinal transport via a prostanoid-mediated mechanism.

Conformational changes couple Na+ and glucose transport

The mechanism by which cotransport proteins couple their substrates across cell membranes is not known. A commonly proposed model is that cotransport results from ligand-induced conformational transitions that change the accessibility of ligand-binding sites from one side of the membrane to the other. To test this model, we have measured the accessibility of covalent probes to a cysteine residue (Q457C) placed in the putative sugar-translocation domain of the Na+/glucose cotransporter (SGLT1). The mutant protein Q457C was able to transport sugar, but transport was abolished after alkylation by methanethiosulfonate reagents. Alkylation blocked sugar translocation but not sugar binding. Accessibility of Q457C to alkylating reagents required external Na+ and was blocked by external sugar and phlorizin. The voltage dependence of accessibility was directly correlated with the presteady–state charge movement of SGLT1. Voltage-jump experiments with rhodamine-6-maleimide-labeled Q457C showed that the time course and level of changes in fluorescence closely followed the presteady–state charge movement. We conclude that conformational changes are responsible for the coupling of Na+ and sugar transport and that Q457 plays a critical role in sugar translocation by SGLT1.