Gestión de dispositivos integrados en una Arquitectura de la Red de las Cosas

La Arquitectura de la Red de las Cosas (IoT) hace referencia a una red de objetos cotidianos interconectados digitalmente. Gracias a IoT, no sólo podemos almacenar, analizar e intercambiar información y datos con dichos objetos, sino que además ellos pueden tener la capacidad de interactuar entre ellos de forma autónoma. Para ellos, los objetos cotidianos disponen de actuadores y sensores que permiten modificar su comportamiento y conocer su estado y propiedades, respectivamente. La gestión de IoT combina todas las funcionalidades necesarias para coordinar un sistema con una Arquitectura de la Red de las Cosas. Una buena gestión del sistema puede reducir costes, mejorar la asistencia a problemas de uso inesperado, corregir fallos y permitir la escalabilidad del sistema permitiéndole la incorporación de nuevos módulos y funcionalidades. En este Proyecto Fin de Grado se realizará primero un análisis de los aspectos de IoT relacionados con la gestión de dispositivos integrados en la Arquitectura de la Red de las Cosas. Después se procederá a realizar la especificación y el diseño de plataforma de gestión. Y finalmente se desarrollarán un caso de uso que permita validar algunos elementos de la plataforma diseñada. Se realizarán distintas pruebas para comprobar una correcta gestión de los dispositivos como el correcto funcionamiento del diseño previamente establecido, por medio, entre otras, de las siguientes operaciones: listar los elementos conectados, posibilidad de obtener y/o modificar dichos elementos (su configuración y su estado) o presentar informes y comprobar el estado en el que se encuentran los dispositivos: operativos o no operativos. De tal forma, en esta memoria se plasma como se ha desarrollado la gestión de dispositivos integrados en un sistema con Arquitectura de la Red de las Cosas utilizando tanto plataformas Intel Galileo como Arduino. ABSTRACT. The Architecture of the Internet of Things (IoT) refers to a network of digitally interconnected everyday objects. With IoT, not only we can store, analyze and exchange information and data with objects, but they can also autonomously interact among them. To accomplish that, the everyday objects are made of actuators and sensors that let us act on their behavior and know their state and properties, respectively. Management of IoT combines all the functionalities needed for coordinating a system with an Architecture of the Internet of Things. A good management system can reduce faults, improve assistance to reduce unexpected problems, correct errors and allow the scalability of the system, allowing the addition of new modules and functionalities. In this Degree Final Project, an analysis about aspects of IoT related to the management of devices integrated into the Architecture of the Internet of things is carried out first. Then, the specification and the design of the management platform is made. Finally, a use case will be developed to validate some elements of the designed platform. Several tests will be run to check the correct management of the devices such as the proper functioning of the design previously established, requesting, among others, the following set of operations: list the connected elements, possibility to obtain or modify these elements (their configuration and their state) or reporting and checking which devices are operating or non-operating. So, in this memory it is explained how it has been carried out the management of devices integrated in a system with an Architecture of the Internet of Things (IoT), based on the Intel Galileo and Arduino platforms.

Fomento del interés por la ciencia y la tecnología para estudiantes de enseñanza secundaria

Las distintas encuestas realizadas sobre la percepción social de la ciencia indican que un alto porcentaje de jóvenes no tienen interés por estas materias. Es muy importante la alfabetización científica de la población ya que ello permite tomar decisiones tanto para la vida cotidiana como sobre distintos problemas que afectan al conjunto de la sociedad. Una sociedad con más conocimiento científico--‐tecnológico lleva asociado un mayor crecimiento económico, pero desafortunadamente nos encontramos con una disminución del número de alumnos que elige estudios de Ciencia y Tecnología. Este desinterés viene influenciado tanto por factores internos al estudiante como por externos. Desde la posición docente podemos actuar sobre ellos promoviendo actividades relacionadas con la divulgación científico--‐tecnológica en clase haciendo uso de medios de comunicación que ofrezcan información procedente de fuentes fiables (prensa escrita, radio, televisión y cibermedios/web 2.0). La propuesta metodológica planteada aquí ayudará a ampliar la alfabetización científico--‐tecnológica a través de la exploración del trabajo de los investigadores actuales y de las biografías de los científicos de otras épocas. Además los alumnos crearán de forma colaborativa material propio para divulgar la Ciencia y la Tecnología en el aula. La propuesta se sitúa en el curso 1º de ESO, pero con perspectivas de extenderla en cursos sucesivos al resto de secundaria y bachillerato adaptando los contenidos . Con este trabajo pretendemos contribuir a incrementar el interés por la Ciencia y la Tecnología en el alumnado de secundaria y bachillerato. ABSTRACT The different surveys conducted in order to analyse the social perception of science show that a high percentage of young people is not interested in these subjects. The level of scientific literacy in the population is very important because it allows people to make their own decisions not only for life management skills but also for coping with the diverse problems that may affect the whole society. A society with more scientific and technological knowledge has attached a higher economic growth, but unfortunately there is a reduction in the number of students that choose Science and Tech studies. This lack of interest is influenced by the student’s internal factors as well as by the external ones. From the teacher’s point of view, it is possible to act on them, promoting activities related to scientific and technological broadcasting in the class, using mass media which can provide information from reliable sources (press, radio, television and media on the Internet/web 2.0) The methodological proposal that is made here will help to broaden scientific literacy through exploring the current researcher’s studies and the former scientifics’ biographies. Students will also create their own material in a collaborative way in order to divulgate science and technology in the classroom. The proposal is set for the first grade of secondary education, but we have in prospect to spread it during the following school years to the rest of secondary education grades and A levels only adapting its content. With this essay we want to contribute to the increasing of the interest in science and technology in high school and A levels students.

Sistema de alarmas asistenciales para hospitales y residencias

En los hospitales y residencias geriátricas de hoy en día es necesario que tengan un sistema asistencial paciente-enfermera. Este sistema debe ser capaz de controlar y gestionar cada una de las alarmas que se puedan generar en el menor tiempo posible y con la mayor eficacia. Para ello se ha diseñado una solución completa llamada ConnectCare. La arquitectura modular del sistema y la utilización de comunicación IP permiten adaptar el sistema a cada situación proporcionando soluciones específicas a medida. Este sistema se compone de un software llamado Buslogic que gestiona las alarmas en un servidor y de unos dispositivos llamados Fonet Control TCP/IP que posee una doble función: por una parte, sirve como dispositivo intercomunicador telefónico y por otra parte, sirve como dispositivo de gestión de alarmas y control de otros dispositivos externos. Como dispositivo intercomunicador telefónico, se integra en la red telefónica como un terminal de extensión analógica permitiendo la intercomunicación entre el paciente y el personal sanitario. Se hará una breve descripción de la parte intercomunicadora pero no es el objeto de este proyecto. En cambio, en la parte de control se hará más hincapié del diseño y su funcionamiento ya que sí es el objeto de este proyecto. La placa de control permite la recepción de señales provenientes de dispositivos de llamadas cableados, como son pulsadores asistenciales tipo “pera” o tiradores de baño. También es posible recibir señales de alerta de dispositivos no estrictamente asistenciales como detectores de humo o detectores de presencia. Además, permite controlar las luces de las habitaciones de los residentes y actuar sobre otros dispositivos externos. A continuación se mostrará un presupuesto para tener una idea del coste que supone. El presupuesto se divide en dos partes, la primera corresponde en el diseño de la placa de control y la segunda corresponde a la fabricación en serie de la misma. Después hablaremos sobre las conclusiones que hemos sacado tras la realización de este proyecto y sobre las posibles mejoras, terminando con una demostración del funcionamiento del equipo en la vida real. ABSTRACT. Nowadays, in hospitals and nursing homes it is required to have a patient-nurse care system. This system must be able to control and manage each one of the alarms, in the shortest possible time and with maximum efficiency. For this, we have designed a complete solution called ConnectCare. The system architecture is modular and the communication is by IP protocol. This allows the system to adapt to each situation and providing specific solutions. This system is composed by a software, called Buslogic, which it manages the alarms in the PC server and a hardware, called Fonet Control TCP / IP, which it has a dual role: the first role, it is a telephone intercom device and second role, it is a system alarm manager and it can control some external devices. As telephone intercom device, it is integrated into the telephone network and also it is an analog extension terminal allowing intercommunication between the patient and the health personnel. A short description of this intercommunication system will be made, because it is not the subject of this project. Otherwise, the control system will be described with more emphasis on the design and operation point of view, because this is the subject of this project. The control board allows the reception of signals from wired devices, such as pushbutton handset or bathroom pullcord. It is also possible to receive warning signals of non nurse call devices such as smoke detectors or motion detectors. Moreover, it allows to control the lights of the patients’ rooms and to act on other external devices. Then, a budget will be showed. The budget is divided into two parts, the first one is related with the design of the control board and the second one corresponds to the serial production of it. Then, it is discussed the conclusions of this project and the possible improvements, ending with a demonstration of the equipment in real life.

El color en la transfiguración del espacio: un estudio empírico de los dispositivos de transformación y configuración

La presente investigación analiza la capacidad del color de cualificar los espacios y propone una clasificación que ordene los diferentes dispositivos que actúan en este proceso. El objetivo de esta tesis es describir estos dispositivos estableciéndolos como parte inequívoca de cualquier proceso desencadenado por la presencia del binomio luz y color. El color ha sido objeto de estudio y experimentación a lo largo de la historia, en todas las artes y las distintas disciplinas relacionadas con la física, la percepción y la interacción. De este modo se realiza una aproximación teórica e histórica al color desde puntos de vista interdisciplinares, para llegar a comprender el modo en el que el color se acaba separando de la forma para pasar a formar parte de la configuración del espacio. Con el propósito de alcanzar este objetivo, se ha realizado un recorrido a través de diferentes teorías y manifestaciones artísticas realizadas en torno al color a lo largo de la Historia, para encontrar las claves de la relación del color con el espacio. Tomando de base este primer acercamiento al tema, se realiza un estudio empírico exhaustivo con modelos físicos con el fin de aislar los dispositivos que intervienen el este proceso en el que el color se pone en relación con el espacio; se analiza su variación a medida que fluctúan las características de los elementos que desencadenan los dispositivos. El objetivo es descubrir un orden, una taxonomía, que permita interpretar cualquier transformación producida por el color en el espacio. Posteriormente, se verifica la validez de esta clasificación estudiando siete espacios modelo en los que el color transfigura el espacio mediante la activación conjunta de varios de los dispositivos analizados. Se ha elegido este grupo de siete espacios debido a que, al tomarlos como suma, sus interiores engloban todos los fenómenos encontrados en el análisis previo. El conjunto de los dispositivos que actúan en cada espacio forma un sistema único e irrepetible que tiene que ver con su transfiguración espacial mediante la luz y el color. Estos sistemas constituyen parte de la génesis propia de cada espacio a través de una secuencia de dispositivos de transformación y configuración espacial que lo hace singular. Los dispositivos clasificados en esta investigación se encuentran en cualquier espacio en el que intervengan la luz y el color, agrupándose secuencialmente en sistemas determinados que cualifican el espacio arquitectónico. ABSTRACT This research analyzes the ability of the colour to qualify the spaces and proposes a classification to arrange the different devices that act in this process. The aim of this thesis is to describe these devices, unequivocally established as part of any process triggered by the presence of light and color pairing. Colour has been the subject of study and experimentation throughout history, in all the arts and disciplines related to physics, perception and interaction. Thus a theoretical and historical approach to the colour is developed from interdisciplinary points of view, in order to understand the way that colour separates from the form and is part of the space configuration. With the purpose of reaching this target, there has been a journey through the artistic movements and theories on colour throughout history, seeking for the key points of its relation with the space. Based on these first intuitions as a starting point, an exhaustive empirical study is made with physical models with the purpose of isolating the devices that participate in this process where colour is related with the space as well as analyzing their variation to the extent that the characteristics of the elements which cause them fluctuate. The objective is to discover an order, a taxonomy that allows to performing any transformation produced by the colour in the space. Afterwards, the validity of this classification is verified by studying seven model spaces in which the colour transfigures the space by the combined activation of several analyzed devices. These seven spaces were chosen due to all the phenomenon set in the previous analysis are included in their interior space. The group of devices that act on each space creates an unrepeatable and unique system that has to do with its spatial transfiguration by means of light and colour. These systems form part of the origin itself of each space through a sequence of devices of spatial transformation and configuration that make this space unique. The devices sorted in this research transform and configure any space in which light and colour participate, sequentially grouping together in determined systems that qualify the architectonic space.

Rapid evolution of sex-related genes in Chlamydomonas

Biological speciation ultimately results in prezygotic isolation—the inability of incipient species to mate with one another–but little is understood about the selection pressures and genetic changes that generate this outcome. The genus Chlamydomonas comprises numerous species of unicellular green algae, including numerous geographic isolates of the species C. reinhardtii. This diverse collection has allowed us to analyze the evolution of two sex-related genes: the mid gene of C. reinhardtii, which determines whether a gamete is mating-type plus or minus, and the fus1 gene, which dictates a cell surface glycoprotein utilized by C. reinhardtii plus gametes to recognize minus gametes. Low stringency Southern analyses failed to detect any fus1 homologs in other Chlamydomonas species and detected only one mid homolog, documenting that both genes have diverged extensively during the evolution of the lineage. The one mid homolog was found in C. incerta, the species in culture that is most closely related to C. reinhardtii. Its mid gene carries numerous nonsynonymous and synonymous codon changes compared with the C. reinhardtii mid gene. In contrast, very high sequence conservation of both the mid and fus1 sequences is found in natural isolates of C. reinhardtii, indicating that the genes are not free to drift within a species but do diverge dramatically between species. Striking divergence of sex determination and mate recognition genes also has been encountered in a number of other eukaryotic phyla, suggesting that unique, and as yet unidentified, selection pressures act on these classes of genes during the speciation process.

Neuronal nicotinic threonine-for-leucine 247 α7 mutant receptors show different gating kinetics when activated by acetylcholine or by the noncompetitive agonist 5-hydroxytryptamine

Mutation of the highly conserved leucine residue (Leu-247) converts 5-hydroxytryptamine (5HT) from an antagonist into an agonist of neuronal homomeric α7 nicotinic acetylcholine receptor expressed in Xenopus oocytes. We show here that acetylcholine (AcCho) activates two classes of single channels with conductances of 44 pS and 58 pS, similar to those activated by 5HT. However, the mean open time of AcCho-gated ion channels (11 ms) is briefer than that of 5HT-gated ion channels (18 ms). Furthermore, whereas the open time of AcCho channels lengthens with hyperpolarization, that of 5HT channels is decreased. In voltage-clamped oocytes, the apparent affinity of the α7 mutant receptor for 5HT is not modified by the presence of dihydro-β-erythroidine, which acts on the AcCho binding site in a competitive manner. This indicates a noncompetitive action of 5HT on nicotinic acetylcholine receptors. Considered together, our findings show that AcCho gates α7 mutant channels with similar conductance but with different kinetic profile than the channels gated by 5HT, suggesting that the two agonists act on different docking sites. These results will help to understand the crosstalk between cholinergic and serotonergic systems in the central nervous system.

Morphological analysis of the mammalian postcranium: A developmental perspective

The past two decades have greatly improved our knowledge of vertebrate skeletal morphogenesis. It is now clear that bony morphology lacks individual descriptive specification and instead results from an interplay between positional information assigned during early limb bud deployment and its “execution” by highly conserved cellular response programs of derived connective tissue cells (e.g., chondroblasts and osteoblasts). Selection must therefore act on positional information and its apportionment, rather than on more individuated aspects of presumptive adult morphology. We suggest a trait classification system that can help integrate these findings in both functional and phylogenetic examinations of fossil mammals and provide examples from the human fossil record.

Glial cell line-derived neurotrophic factor promotes the development of adrenergic neurons in mouse neural crest cultures

Growth of mouse neural crest cultures in the presence of glial cell line-derived neurotrophic factor (GDNF) resulted in a dramatic dose-dependent increase in the number of tyrosine hydroxylase (TH)-positive cells that developed when 5% chicken embryo extract was present in the medium. In contrast, growth in the presence of bone morphogenetic protein (BMP)-2, BMP-4, BMP-6, transforming growth factor (TGF) β1, TGF-β2, and TGF-β3 elicited no increase in the number of TH-positive cells. The TH-positive cells that developed in the presence of GDNF had neuronal morphology and contained the middle and low molecular weight neurofilament proteins. Numerous TH-negative cells with the morphology of neurons also were observed in GDNF-treated cultures. Analysis revealed that the period from 6 to 12 days in vitro was the critical time for exposure to GDNF to generate the increase in TH-positive cell number. The growth factors neurotrophin-3 and fibroblast growth factor-2 elicited increases in the number of TH-positive cells similar to that seen in response to GDNF. In contrast, nerve growth factor was unable to substitute for GDNF. These findings extend the previously reported biological activities of GDNF by showing that it can act on mouse neural crest cultures to promote the development of neurons.

RGS2/G0S8 is a selective inhibitor of Gqα function

RGS (regulators of G protein signaling) proteins are GTPase activating proteins that inhibit signaling by heterotrimeric G proteins. All RGS proteins studied to date act on members of the Giα family, but not Gsα or G12α. RGS4 regulates Giα family members and Gqα. RGS2 (G0S8) is exceptional because the G proteins it regulates have not been identified. We report that RGS2 is a selective and potent inhibitor of Gqα function. RGS2 selectively binds Gqα, but not other Gα proteins (Gi, Go, Gs, G12/13) in brain membranes; RGS4 binds Gqα and Giα family members. RGS2 binds purified recombinant Gqα, but not Goα, whereas RGS4 binds either. RGS2 does not stimulate the GTPase activities of Gsα or Giα family members, even at a protein concentration 3000-fold higher than is sufficient to observe effects of RGS4 on Giα family members. In contrast, RGS2 and RGS4 completely inhibit Gq-directed activation of phospholipase C in cell membranes. When reconstituted with phospholipid vesicles, RGS2 is 10-fold more potent than RGS4 in blocking Gqα-directed activation of phospholipase Cβ1. These results identify a clear physiological role for RGS2, and describe the first example of an RGS protein that is a selective inhibitor of Gqα function.

MAL, an Integral Element of the Apical Sorting Machinery, Is an Itinerant Protein That Cycles between the Trans-Golgi Network and the Plasma Membrane

The MAL proteolipid is a nonglycosylated integral membrane protein found in glycolipid-enriched membrane microdomains. In polarized epithelial Madin-Darby canine kidney cells, MAL is necessary for normal apical transport and accurate sorting of the influenza virus hemagglutinin. MAL is thus part of the integral machinery for glycolipid-enriched membrane–mediated apical transport. At steady state, MAL is predominantly located in perinuclear vesicles that probably arise from the trans-Golgi network (TGN). To act on membrane traffic and to prevent their accumulation in the target compartment, integral membrane elements of the protein-sorting machinery should be itinerant proteins that cycle between the donor and target compartments. To establish whether MAL is an itinerant protein, we engineered the last extracellular loop of MAL by insertion of sequences containing the FLAG epitope or with sequences containing residues that became O-glycosylated within the cells or that displayed biotinylatable groups. The ectopic expression of these modified MAL proteins allowed us to investigate the surface expression of MAL and its movement through different compartments after internalization with the use of a combination of assays, including surface biotinylation, surface binding of anti-FLAG antibodies, neuraminidase sensitivity, and drug treatments. Immunofluorescence and flow cytometric analyses indicated that, in addition to its Golgi localization, MAL was also expressed on the cell surface, from which it was rapidly internalized. This retrieval implies transport through the endosomal pathway and requires endosomal acidification, because it can be inhibited by drugs such as chloroquine, monensin, and NH4Cl. Resialylation experiments of surface MAL treated with neuraminidase indicated that ∼30% of the internalized MAL molecules were delivered to the TGN, probably to start a new cycle of cargo transport. Together, these observations suggest that, as predicted for integral membrane members of the late protein transport machinery, MAL is an itinerant protein cycling between the TGN and the plasma membrane.

Long RNA hairpins that contain inosine are present in Caenorhabditis elegans poly(A)+ RNA

Adenosine deaminases that act on RNA (ADARs) are RNA-editing enzymes that convert adenosine to inosine within double-stranded RNA. In the 12 years since the discovery of ADARs only a few natural substrates have been identified. These substrates were found by chance, when genomically encoded adenosines were identified as guanosines in cDNAs. To advance our understanding of the biological roles of ADARs, we developed a method for systematically identifying ADAR substrates. In our first application of the method, we identified five additional substrates in Caenorhabditis elegans. Four of those substrates are mRNAs edited in untranslated regions, and one is a noncoding RNA edited throughout its length. The edited regions are predicted to form long hairpin structures, and one of the RNAs encodes POP-1, a protein involved in cell fate decisions.

Abolition of morphine-immunosuppression in mice lacking the μ-opioid receptor gene

Opiates are potent analgesic and addictive compounds. They also act on immune responses, and morphine, the prototypic opiate, has been repeatedly described as an immunosuppressive drug. Pharmacological studies have suggested that the inhibitory action of opiates on immunity is mediated by multiple opioid receptor sites but molecular evidence has remained elusive. Recently, three genes encoding μ- (MOR), δ-, and κ-opioid receptors have been cloned. To investigate whether the μ-opioid receptor is functionally implicated in morphine immunosuppression in vivo, we have examined immune responses of mice with a genetic disruption of the MOR gene. In the absence of drug, there was no difference between wild-type and mutant mice with regard to a large number of immunological endpoints, suggesting that the lack of MOR-encoded protein has little consequence on immune status. Chronic morphine administration induced lymphoid organ atrophy, diminished the ratio of CD4+CD8+ cells in the thymus and strongly reduced natural killer activity in wild-type mice. None of these effects was observed in MOR-deficient mice after morphine treatment. This demonstrates that the MOR gene product represents a major molecular target for morphine action on the immune system. Because our previous studies of MOR-deficient mice have shown that this receptor protein is also responsible for morphine analgesia, reward, and physical dependence, the present results imply that MOR-targeted therapeutic drugs that are developed for the treatment of pain or opiate addiction may concomitantly influence immune responses.

Preferential synaptic relationships between substance P-immunoreactive boutons and neurokinin 1 receptor sites in the rat spinal cord

Substance P plays an important role in the transmission of pain-related information in the dorsal horn of the spinal cord. Recent immunocytochemical studies have shown a mismatch between the distribution of substance P and its receptor in the superficial laminae of the dorsal horn. Because such a mismatch was not observed by using classical radioligand binding studies, we decided to investigate further the issue of the relationship between substance P and its receptor by using an antibody raised against a portion of the carboxyl terminal of the neurokinin 1 receptor and a bispecific monoclonal antibodies against substance P and horseradish peroxidase. Light microscopy revealed a good correlation between the distributions of substance P and the neurokinin 1 receptor, both being localized with highest densities in lamina I and outer lamina II of the spinal dorsal horn. An ultrastructural double-labeling study, combining preembedding immunogold with enzyme-based immunocytochemistry, showed that most neurokinin 1 receptor immunoreactive dendrites were apposed by substance P containing boutons. A detailed quantitative analysis revealed that neurokinin 1 receptor immunoreactive dendrites received more appositions and synapses from substance P immunoreactive terminals than those not expressing the neurokinin 1 receptor. Such preferential innervation by substance P occurred in all superficial dorsal horn laminae even though neurokinin 1 receptor immunoreactive dendrites were a minority of the total number of dendritic profiles in the above laminae. These results suggest that, contrary to the belief that neuropeptides act in a diffuse manner at a considerable distance from their sites of release, substance P should act on profiles expressing the neurokinin 1 receptor at a short distance from its site of release.

Pectate lyase A, an enzymatic subunit of the Clostridium cellulovorans cellulosome

Clostridium cellulovorans uses not only cellulose but also xylan, mannan, pectin, and several other carbon sources for its growth and produces an extracellular multienzyme complex called the cellulosome, which is involved in plant cell wall degradation. Here we report a gene for a cellulosomal subunit, pectate lyase A (PelA), lying downstream of the engY gene, which codes for cellulosomal enzyme EngY. pelA is composed of an ORF of 2,742 bp and encodes a protein of 914 aa with a molecular weight of 94,458. The amino acid sequence derived from pelA revealed a multidomain structure, i.e., an N-terminal domain partially homologous to the C terminus of PelB of Erwinia chrysanthemi belonging to family 1 of pectate lyases, a putative cellulose-binding domain, a catalytic domain homologous to PelL and PelX of E. chrysanthemi that belongs to family 4 of pectate lyases, and a duplicated sequence (or dockerin) at the C terminus that is highly conserved in enzymatic subunits of the C. cellulovorans cellulosome. The recombinant truncated enzyme cleaved polygalacturonic acid to digalacturonic acid (G2) and trigalacturonic acid (G3) but did not act on G2 and G3. There have been no reports available to date on pectate lyase genes from Clostridia.

Yeast Rab GTPase-activating Protein Gyp1p Localizes to the Golgi Apparatus and Is a Negative Regulator of Ypt1p

A family of related proteins in yeast Saccharomyces cerevisiae is known to have in vitro GTPase-activating protein activity on the Rab GTPases. However, their in vivo function remains obscure. One of them, Gyp1p, acts on Sec4p, Ypt1p, Ypt7p, and Ypt51p in vitro. Here, we present data to reveal its in vivo substrate and the role that it plays in the function of the Rab GTPase. Red fluorescent protein-tagged Gyp1p is concentrated on cytoplasmic punctate structures that largely colocalize with a cis-Golgi marker. Subcellular fractionation of a yeast lysate confirmed that Gyp1p is peripherally associated with membranes and that it cofractionates with Golgi markers. This localization suggests that Gyp1p may only act on Rab GTPases on the Golgi. A gyp1Δ strain displays a growth defect on synthetic medium at 37°C. Overexpression of Ypt1p, but not other Rab GTPases, strongly inhibits the growth of gyp1Δ cells. Conversely, a partial loss-of-function allele of YPT1, ypt1-2, can suppress the growth defect of gyp1Δ cells. Furthermore, deletion of GYP1 can partially suppress growth defects associated with mutants in subunits of transport protein particle complex, a complex that catalyzes nucleotide exchange on Ypt1p. These results establish that Gyp1p functions on the Golgi as a negative regulator of Ypt1p.