Criterios técnicos del proyecto con módulos tridimensionales ligeros: las casas del Solar Decathlon 2005 y 2007

Este trabajo se ocupa de la construcción modular ligera de viviendas unifamiliares, más concretamente aborda el problema de la definición constructiva de las mismas, habida cuenta de lo poco conocido y publicado de este aspecto en particular. Dado que la construcción es fruto de la evolución, resulta de gran importancia el estudio de los antecedentes para comprender la situación actual, por ello para documentar como corresponde el estado de la técnica y de la investigación se estudiaron los antecedentes desde sus primeras manifestaciones a finales del siglo XIX hasta nuestros días. Contrariamente a las profecías de Le Corbusier y otros, la industrialización no ha llegado a la construcción ni en la forma ni en la medida que era de esperar a principios del siglo XX. Sin embargo y a pesar del relativo “fracaso” de la industrialización en convertirse en la forma mayoritaria de producir edificios, lo cierto es que algunos sistemas altamente industrializados, como lo es la construcción modular ligera, han logrado un lugar en el mercado, que en algunos países resulta cuanto menos significativo. Delimitar el estado de la técnica y la situación actual es crucial en este trabajo, toda vez que la construcción industrializada la realizan empresas, y situarse al margen de las mismas y de su producción, nos guste o no, resulta contrario al sentido común. De este modo se han identificado y documentado ejemplos como el de EE.UU. y Japón, entre otros, que resultan muy ilustrativos tanto desde la evolución como del estado actual. Del estado de la técnica y la investigación también resulta la escasez de publicación de detalles constructivos específicos de la construcción modular ligera. Es obvio que los fabricantes intentan blindarse mediante el secreto industrial, logrando que las soluciones constructivas más interesantes queden fuertemente protegidas dentro del conocimiento reservado de la empresa. De este modo un sistema que alcanza grados de prefabricación del 95%, tan atractivo e interesante para el arquitecto queda reservado al conocimiento de unos pocos dentro de las empresas. La búsqueda de mayor información sobre estos sistemas encuentra un filón indiscutible en los proyectos de las casas del concurso Solar Decathlon. En efecto, debido a las condiciones particulares de este concurso, las casas que compiten deben ser instaladas en un plazo corto, por lo que en su mayoría recurren al método de construcción modular ligera, para lograr correctamente este objetivo. Adicionalmente todos los planos de proyecto incluidos los de detalle, así como memorias de especificaciones técnicas, quedan colgadas en la Web, a libre disposición del público. De este modo se ha considerado interesante, conveniente y útil, aprovechar como fuente documental de este trabajo, aparte de lo obtenido en el estado de la técnica y la investigación, los proyectos de las casas de los concursos 2005 y 2007, que fueron las dos últimas ediciones celebradas cuando se inició esta tesis. De modo similar a esta carencia de detalles publicados se observa igualmente una falta de investigación y de metodologías adecuadas a la misma sobre soluciones y detalles constructivos propios de la construcción modular ligera. Por eso y ante la dificultad de manejar adecuadamente una información disponible pero farragosa, este trabajo ha dedicado una parte importante de su esfuerzo a la creación de una metodología adecuada a este tipo de situación. Hay que destacar que cada proyecto puede constar fácilmente de 60 a casi 200 planos, sin contar las memorias técnicas y otros documentos, tales como galerías de imágenes, etc. Por otra parte resulta útil establecer esta metodología, no solo para esta tesis, sino para futuros trabajos de investigación sobre el tema, toda vez que con posterioridad a 2007 se han celebrado nuevas ediciones del concurso con su correspondiente volumen de información disponible. La metodología del análisis de los proyectos se basa en el diseño y creación de siete fichas tipo que resumen los aspectos fundamentales del proyecto desde el punto de vista constructivo, permitiendo de este modo su rápida visualización y comprensión sin pretender exhaustividad, ya que en caso de querer profundizar en el detalle de la información siempre está el proyecto original para ello. Tras estudiar la información obtenida de los proyectos de las casas de concurso, se contrastan y discuten los resultados para obtener conocimientos de interés para el objetivo propuesto en la tesis. Se comparan los resultados procedentes del estado de la técnica y de la investigación y se obtienen las conclusiones correspondientes. De este modo ha sido posible identificar una serie de criterios técnicos de proyecto de viviendas unifamiliares realizadas mediante construcción modular ligera, que además se organizan en varios niveles, por lo que el resultado es un conjunto de criterios como germen de una futura guía o manual. Como conclusiones fundamentales de la tesis hay que destacar las metodológicas, que habilitan la extensión de este estudio a otros trabajos y la aportación original al conocimiento con la definición de una serie de criterios técnicos de proyecto de viviendas realizadas mediante construcción modular ligera, que además de mejorar esta parte del saber constructivo serán de gran ayuda a los arquitectos de cara a la reducción de errores, que a menudo derivan en mayores costes y plazos, cuando no directamente al abandono del sistema constructivo. El trabajo de la tesis se estructura en siete capítulos a saber: Capítulo 1: Introducción, donde se explica el tema de la tesis, el objetivo principal, las limitaciones y se aportan algunas definiciones. Capítulo 2: Estado de la técnica y la investigación, dentro del cual se presentan los antecedentes desde sus inicios pasando por la primera y segunda mitad del siglo XX y finalizando con el estado actual que recorre los sistemas de EE.UU, Japón, Europa, Escandinavia, centro Europa, Francia, España y algunos ejemplos de Latinoamérica. En el estado de la investigación se presentan las publicaciones tanto en revistas científicas indexadas en el JCR, como publicaciones no indexadas en las que se muestran artículos científicos, comunicaciones a congresos, documentación sobre el concurso Solar Decathlon, libros específicos y libros genéricos sobre construcción modular ligera, tesis doctorales tanto generales como específicas sobre el concurso Solar Decathlon, documentos de idoneidad técnica, páginas Web del concurso Solar Decathlon y finalmente páginas Web de empresas de construcción modular ligera y patentes. También se expresan las conclusiones parciales del capítulo así también como la justificación y los objetivos particulares. Capítulo 3: Metodología, se expone en primer lugar el planteamiento del problema, para luego desarrollar los métodos utilizados para el estudio y clasificación del transporte y de la organización modular, la relación entre la organización modular y distribución espacial, el diseño y contenido de las fichas resumen de los 38 proyectos presentados al SD2005 y SD2007, así también como la información consultada para su realización. Se explica asimismo como se realizaron las tablas comparativas partiendo de la información de las fichas. Finalmente se expresa la manera en que se trató el estudio particular de la junta entre módulos. Capítulo 4: Discusión de resultados, dónde en primer lugar se presenta la clasificación y las estrategias del transporte así también como una clasificación de la organización modular, en segundo lugar se estudian las relaciones que existen entre el sistema modular y la organización espacial. A partir de aquí se muestra el estudio de la estructura de todas las casas del SD2005 y SD2007, según el tipo y material de las zapatas, mostrando tanto tablas cuantitativas como tablas gráficas con fotos. De la misma manera se tratan los forjados, los pilares y las vigas y las cubiertas. Se estudia además el cerramiento opaco y acristalado, la cubierta y el forjado según las capas que lo componen. En el caso de las instalaciones se estudian las que son especiales, descartando las normales en este tipo de casas, como son los paneles fotovoltaicos, placas térmicas o tubos de vacío y los sistemas de almacenamiento de energía, como las baterías, presentando tablas numéricas y gráficas con fotos. El transporte se analiza según corresponda al traslado de la vivienda o al transporte de apoyo, según el tipo y la cantidad utilizado. Con respecto al montaje se diferencia en si se usó grúa o no, y en el caso de no utilizarse se muestran los métodos alternativos. Con respecto a la exploración de la organización modular se presentan la cantidad de módulos que utilizó cada casa, así como también la cuantificación de los sistemas híbridos como son elementos lineales 1D, paneles 2D. Por último se muestra el estudio detallado de la junta entre módulos 3D. Finalmente se realiza la propuesta de un conjunto de criterios técnicos de proyecto organizado en cuatro niveles: 1º nivel de criterios generales, 2º nivel sobre sistemas constructivos, 3º nivel de detalles constructivos y el 4º nivel llamado logística obras previas, transporte y montaje. Capítulo 5: Conclusiones y líneas futuras de investigación, se exponen las conclusiones generales, metodológicas, documentales y por último las de construcción modular. Finalmente se realiza una propuesta de líneas futuras de investigación. Capítulo 6: Bibliografía. Capítulo 7: Anexos, en el que se presentan todas las fichas resumen de las casas realizadas por el autor de esta tesis. Además se incluyen los casos concretos de Voisin en Francia y la TVA (Tennessee Valley Authority) en EE.UU., así como referencias de manuales genéricos de construcción ligera, medidas de transporte en España e información sobre estadísticas de viviendas en la Unión Europea. ABSTRACT This paper deals with Light Modular Construction of houses, and specifically addresses the problem of the constructive definition of the same, given how little known and published this subject has been. Since building construction is the result of evolution, it is of great importance to study its background for understanding the current situation, therefore to document the state of the art and research, its history was studied from its origins dating to the end nineteenth century to our days. Contrary to the prophecies of Le Corbusier and others, industrialization has not reached the construction neither in the form nor to the extent that it was expected in the early twentieth century. However, despite the relative "failure" of industrialization to become the major form of production for buildings, the fact is that some highly industrialized systems, such as the so called Lightweight Modular Construction, have achieved a place in the market, which in some countries is at least significant. To outline the state of the art and the current situation is crucial in this work, since industrialized construction is carried out by companies, and to step away from them and their production, whether we like it or not, it is contrary to common sense. So that, several cases have been identified and documented, such as the US and Japan examples, among others, which are very illustrative both from evolution and the current status. The state of the art and research shows also a shortage of publication of specific construction details of light modular construction. Obviously, manufacturers try to shield themselves by trade secret, making the most interesting constructive solutions remain heavily protected within the reserved knowledge of the company, so a system that reaches levels of 95% prefabrication, so attractive and interesting for the architect It is reserved to the knowledge of a few people inside the companies. The search for more information on these systems finds an invaluable reef in the projects of the Solar Decathlon houses. Indeed, due to the particular conditions of this contest, houses competing must be installed in a short time, so mostly turn to modular construction methods for this purpose to achieve properly this goal. Additionally all levels of project, including detailed and technical specifications reports are published on the Web, freely available to the general public. Thus it was considered interesting, convenient and useful to take advantage as a documentary source of this work, apart from what it were obtained in the state of the art and research, the projects of the houses of the 2005 and 2007 contests, which were the last two editions already held before this thesis was started. Similar to this lack of published details it is also observed a lack of research and methodologies adapted to the solutions and construction details of lightweight modular construction. So that, and facing the difficulty of adequately manage the available but bulky information, this work has devoted a significant part of its effort to create an appropriate methodology for this type of situation. It is noteworthy that each project can easily consist of 60 to almost 200 blueprints, not including technical reports and other documents, such as photo galleries, etc. The methodology of the analysis of the projects is based on the design and creation of seven record sheets that summarize key aspects of the project from a construction point of view, thus allowing quick watching and understanding of the project, without claiming completeness, always keeping for further information the blueprints themselves. After studying the information obtained from the projects of the competition houses, the results are compared and discussed to obtain relevant knowledge according to the objective proposed in the thesis. The results from the state of the art and research are also compared and the conclusions so obtained make possible to identify a number of technical design criteria for single family homes made using lightweight modular construction, which also have been organized at various levels, so the result is a set of criteria as a seed for a future guide or manual. Among the main conclusions of the thesis must be noted the methodological ones, that enable the extension of this study to other works and are an original contribution to knowledge with the definition of a number of technical criteria for housing projects made by light modular construction, what in addition to improving this part of the constructive knowledge will be of great help for the architects to reduce errors that often result in higher costs and delays, if not in abandonment of the construction system itself. The work of the thesis is divided into seven chapters as follows: Chapter 1: Introduction, where the topic of the thesis, the main objective, limitations and the provided definitions is explained. Chapter 2: State of the art and research, within which history is presented from the beginning through the first and second half of the twentieth century and ending with the current systems, that spans from the USA to Japan, Europe, Scandinavia, Central Europe, France, Spain and some Latin American examples. In the state of research publications are presented both on scientific journals indexed in the JCR, and non-indexed publications in which scientific articles, conference papers, information about the Solar Decathlon competition, generic and specific books on light modular construction, articles both general and specific on the Solar Decathlon competition, technical approval documents, dissertations, the Solar Decathlon Web pages and finally lightweight modular construction companies Web pages and patents. The partial conclusions of the chapter as well as the rationale and specific objectives are also expressed. Chapter 3: Methodology first of all exposes the problem statement and then develops the methods used for the study and classification of transportation and modular organization, the relationship between the modular organization and spatial distribution, design and content summary records of the 38 projects submitted to the SD2005 and SD2007, as well as information consulted for its realization. It explains equally how the comparative tables based on information from the chips were made. Finally, the way the particular study of the joint between modules is carried out is also treated. Chapter 4: Discussion, first sorting a classification of transport strategies as well as of modular organization, secondly the relationship between the modular system and the spatial organization studied is presented. From then on, the study of the structure of every house Moreover, to establish this methodology is useful not only for this thesis, but for future research on the subject, since after 2007 there have been held new editions of the contest with a corresponding volume of information available. shown in the SD2005 and SD2007 contests, depending on the type and material of the shoe, showing both quantitative tables as graphic boards with pictures is carried out. Similarly slabs, columns and beams and roofs are treated. Furthermore, the opaque and transparent façades, as well as roof and floor enclosure, according to the layers that compose them are studied. In the case of facilities there have been only addressed that which are of a special type, discarding that considered usual in this type of houses such as photovoltaic panels, thermal panels or vacuum tubes and energy storage systems such as batteries, presenting numerical and graphical tables with photos .The transportation is analyzed depending on it is used to move the house or for additional support, depending on the type and quantity of items used. Regarding assembly it has been made a difference if crane is or not used and if not, what the alternative methods are. With respect to the exploration of the modular organization, the amount of modules used in each house is presented, as well as the quantification of hybrid systems such as linear members as well as 1D, 2D panels. Finally, it has been carried out the detailed study of the joint between 3D modules. Finally it is proposed a set of technical criteria organized into four levels, 1st level of general criteria, 2nd level on building systems, 3rd level construction details and 4th level called previous works and logistics, transportation and assembly. Chapter 5: Conclusions and future research, where general, methodological, documentary and finally modular construction findings are presented. A proposal for further research is done. Chapter 6: Bibliography. Chapter 7: Annexes, in which all the record sheets of houses made by the author of this thesis are presented. Besides the specific cases of Voisin in France and TVA (Tennessee Valley Authority) in the USA, as well as general reference manuals on lightweight construction, transportation dimensions in Spain, and information on housing statistics in the European Union are included.

Involvement of protein kinase A in fibroblast growth factor-2-activated transcription

Polypeptide growth factors activate common signal transduction pathways, yet they can induce transcription of different target genes. The mechanisms that control this specificity are not completely understood. Recently, we have described a fibroblast growth factor (FGF)-inducible response element, FiRE, on the syndecan-1 gene. In NIH 3T3 cells, the FiRE is activated by FGF-2 but not by several other growth factors, such as platelet-derived growth factor or epidermal growth factor, suggesting that FGF-2 activates signaling pathways that diverge from pathways activated by other growth factors. In this paper, we report that the activation of FiRE by FGF-2 requires protein kinase A (PKA) in NIH 3T3 cells. The PKA-specific inhibitor H-89 (N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide) blocked the FGF-2-induced activation of FiRE, the transcription of the syndecan-1 gene, and cell proliferation. Also, expression of a dominant-negative form of PKA inhibited the FGF-2-induced FiRE activation and the transcription of the syndecan-1 gene. The binding of activator protein-1 transcription-factor complexes, required for the activation of FiRE, was blocked by inhibition of PKA activity before FGF-2 treatment. In accordance with the growth factor specificity of FiRE, the activity of PKA was stimulated by FGF-2 but not by platelet-derived growth factor or epidermal growth factor. Furthermore, a portion of the PKA catalytic subunit pool was translocated to the nucleus by FGF-2. Noticeably, the total cellular cAMP concentration was not affected by FGF-2 stimulus. We propose that the FGF-2-selective transcriptional activation through FiRE is caused by the ability of FGF-2 to control PKA activity.

Interaction with mLin-7 Alters the Targeting of Endocytosed Transmembrane Proteins in Mammalian Epithelial Cells

To investigate the targeting mechanism for proteins bound to the mammalian Lin-7 (mLin-7) PDZ domain, we created receptor protein chimeras composed of the carboxyl-terminal amino acids of LET-23 fused to truncated nerve growth factor receptor/P75. mLin-7 bound to the chimera with a wild-type LET-23 carboxyl-terminal tail (P75t-Let23WT), but not a mutant tail (P75t-Let23MUT). In Madin-Darby canine kidney (MDCK) cells, P75t-Let23WT localized to the basolateral plasma membrane domain, whereas P75t-Let23MUT remained apical. Furthermore, mutant mLin-7 constructs acted as dominant interfering proteins and inhibited the basolateral localization of P75t-Let23WT. The mechanisms for this differential localization were examined further, and, initially, we found that P75t-Let23WT and P75t-Let23MUT were delivered equally to the apical and basolateral plasma membrane domains. Although basolateral retention of P75t-Let23WT, but not P75t-Let23MUT, was observed, the greatest difference in receptor localization was seen in the rapid trafficking of P75t-Let23WT to the basolateral plasma membrane domain after endocytosis, whereas P75t-Let23MUT was degraded in lysosomes, indicating that mLin-7 binding can alter the fate of endocytosed proteins. Altogether, these data support a model for basolateral protein targeting in mammalian epithelial cells dependent on protein–protein interactions with mLin-7, and also suggest a dynamic role for mLin-7 in endosomal sorting.

Phosphorylated Ser/Arg-rich proteins: limiting factors in the assembly of 200S large nuclear ribonucleoprotein particles.

We have previously shown that specific nuclear pre-mRNA transcripts and their splicing products, as well as the general population of nuclear poly(A)+ RNA, are packaged in large nuclear ribonucleoprotein (InRNP) particles that sediment at the 200S region in sucrose gradients. The InRNP particles contain all uridine-rich small nuclear ribonucleoprotein complexes required for pre-mRNA splicing, as well as protein splicing factors. In this paper we show that all of the phosphorylated, mAb 104 detectable, Ser/Arg-rich essential splicing factors (SR proteins) in the nucleoplasm are integral components of the InRNP particles, whereas only part of the essential splicing factor U2AF65 (U2 snRNP auxiliary factor) and the polypyrimidine tract binding protein (PTB) are associated with these particles. This finding suggests a limiting role for SR proteins in the assembly of the InRNP particles. We further show that the structural integrity of InRNP particles is sensitive to variations in the phosphorylation levels of the SR proteins.

A novel role for retromer in the control of epithelial cell polarity

The establishment and maintenance of epithelial cell polarity is essential throughout the development and adult life of all multicellular organisms. A key player in maintaining epithelial polarity is Crumbs (Crb), an evolutionarily conserved type-I transmembrane protein initially identified in Drosophila. Correct Crb levels and apical localization are imperative for its function. However, as is the case for many polarized proteins, the mechanisms of its trafficking and strict apical localization are poorly understood. To address these questions, we developed a liposome-based assay to identify trafficking coats and interaction partners of Crb in a native-like environment. Thereby, we demonstrated that Crb is a cargo for Retromer, a trafficking complex required for transport from endosomes to the trans-Golgi-network. The functional importance of this interaction was revealed by studies in Drosophila epithelia, which established Retromer as a novel regulator of epithelial cell polarity and verified the vast potential of this technique.

Grain size effects on excess Thorium-230 of sediment cores from the Southern Ocean and the South East Atlantic

Excess Thorium-230 (230Thxs) as a constant flux tracer is an essential tool for paleoceanographic studies, but its limitations for flux normalization are still a matter of debate. In regions of rapid sediment accumulation, it has been an open question if 230Thxs-normalized fluxes are biased by particle sorting effects during sediment redistribution. In order to study the sorting effect of sediment transport on 230Thxs, we analyzed the specific activity of 230Thxs in different particle size classes of carbonate-rich sediments from the South East Atlantic, and of opal-rich sediments from the Atlantic sector of the Southern Ocean. At both sites, we compare the 230Thxs distribution in neighboring high vs. low accumulation settings. Two grain-size fractionation methods are explored. We find that the 230Thxs distribution is strongly grain size dependent, and 50-90% of the total 230Thxs inventory is concentrated in fine material smaller than 10 µm, which is preferentially deposited at the high accumulation sites. This leads to an overestimation of the focusing factor Psi, and consequently to an underestimation of the vertical flux rate at such sites. The distribution of authigenic uranium indicates that fine organic-rich material has also been re-deposited from lateral sources. If the particle sorting effect is considered in the flux calculations, it reduces the estimated extent of sediment focusing. In order to assess the maximum effect of particle sorting on Psi, we present an extreme scenario, in which we assume a lateral sediment supply of only fine material (< 10 µm). In this case, the focusing factor of the opal-rich core would be reduced from Psi = 5.9 to Psi = 3.2. In a more likely scenario, allowing silt-sized material to be transported, Psi is reduced from 5.9 to 5.0 if particle sorting is taken into consideration. The bias introduced by particle sorting is most important for strongly focused sediments. Comparing 230Thxs-normalized mass fluxes biased by sorting effects with uncorrected mass fluxes, we suggest that 230Thxs-normalization is still a valid tool to correct for lateral sediment redistribution. However, differences in focusing factors between core locations have to be evaluated carefully, taking the grain size distributions into consideration.

Structural characterization of interaction of K11 and K63 mixed-linkage polyubiquitin chains with the tUIMs of epsin1

Ubiquitylation or covalent attachment of ubiquitin (Ub) to a variety of substrate proteins in cells is a versatile post-translational modification involved in the regulation of numerous cellular processes. The distinct messages that polyubiquitylation encodes are attributed to the multitude of conformations possible through attachment of ubiquitin monomers within a polyubiquitin chain via a specific lysine residue. Thus the hypothesis is that linkage defines polyubiquitin conformation which in turn determines specific recognition by cellular receptors. Ubiquitylation of membrane surface receptor proteins plays a very important role in regulating receptor-mediated endocytosis as well as endosomal sorting for lysosomal degradation. Epsin1 is an endocytic adaptor protein with three tandem UIMs (Ubiquitin Interacting Motifs) which are responsible for the highly specific interaction between epsin and ubiquitylated receptors. Epsin1 is also an oncogenic protein and its expression is upregulated in some types of cancer. Recently it has been shown that novel K11 and K63 mixed-linkage polyubiquitin chains serve as internalization signal for MHC I (Major Histocompatibility Complex I) molecule through their association with the tUIMs of epsin1. However the molecular mode of action and structural details of the interaction between polyubiquitin chains on receptors and tUIMs of epsin1 is yet to be determined. This information is crucial for the development of anticancer therapeutics targeting epsin1. The molecular basis for the linkage-specific recognition of K11 and K63 mixed-linkage polyubiquitin chains by the tandem UIMs of the endocytic adaptor protein epsin1 is investigated using a combination of NMR methods.

Clathrin adaptor epsinR is required for retrograde sorting on early endosomal membranes

Retrograde transport links early/recycling endosomes to the trans-Golgi network (TGN), thereby connecting the endocytic and the biosynthetic/secretory pathways. To determine how internalized molecules are targeted to the retrograde route, we have interfered with the function of clathrin and that of two proteins that interact with it, AP1 and epsinR. We found that the glycosphingolipid binding bacterial Shiga toxin entered cells efficiently when clathrin expression was inhibited. However, retrograde transport of Shiga toxin to the TGN was strongly inhibited. This allowed us to show that for Shiga toxin, retrograde sorting on early/recycling endosomes depends on clathrin and epsinR, but not AP1. EpsinR was also involved in retrograde transport of two endogenous proteins, TGN38/46 and mannose 6-phosphate receptor. In conclusion, our work reveals the existence of clathrin-independent and -dependent transport steps in the retrograde route, and establishes a function for clathrin and epsinR at the endosome-TGN interface.

The Yeast GRD20 Gene Is Required for Protein Sorting in the trans-Golgi Network/Endosomal System and for Polarization of the Actin Cytoskeleton

The proper localization of resident membrane proteins to the trans-Golgi network (TGN) involves mechanisms for both TGN retention and retrieval from post-TGN compartments. In this study we report identification of a new gene, GRD20, involved in protein sorting in the TGN/endosomal system of Saccharomyces cerevisiae. A strain carrying a transposon insertion allele of GRD20 exhibited rapid vacuolar degradation of the resident TGN endoprotease Kex2p and aberrantly secreted ∼50% of the soluble vacuolar hydrolase carboxypeptidase Y. The Kex2p mislocalization and carboxypeptidase Y missorting phenotypes were exhibited rapidly after loss of Grd20p function in grd20 temperature-sensitive mutant strains, indicating that Grd20p plays a direct role in these processes. Surprisingly, little if any vacuolar degradation was observed for the TGN membrane proteins A-ALP and Vps10p, underscoring a difference in trafficking patterns for these proteins compared with that of Kex2p. A grd20 null mutant strain exhibited extremely slow growth and a defect in polarization of the actin cytoskeleton, and these two phenotypes were invariably linked in a collection of randomly mutagenized grd20 alleles. GRD20 encodes a hydrophilic protein that partially associates with the TGN. The discovery of GRD20 suggests a link between the cytoskeleton and function of the yeast TGN.

Rab9A is required for delivery of cargo from recycling endosomes to melanosomes

Melanosomes are a type of lysosome-related organelle that is commonly defective in Hermansky-Pudlak syndrome. Biogenesis of melanosomes is regulated by BLOC-1, -2, -3, or AP-1, -3 complexes, which mediate cargo transport from recycling endosomes to melanosomes. Although several Rab GTPases have been shown to regulate these trafficking steps, the precise role of Rab9A remains unknown. Here, we found that a cohort of Rab9A associates with the melanosomes and its knockdown in melanocytes results in hypopigmented melanosomes due to mistargeting of melanosomal proteins to lysosomes. In addition, the Rab9A-depletion phenotype resembles Rab38/ 32-inactivated or BLOC-3-deficient melanocytes, suggesting that Rab9A works in line with BLOC-3 and Rab38/ 32 during melanosome cargo transport. Furthermore, silencing of Rab9A, Rab38/ 32 or its effector VARP, or BLOC-3-deficiency in melanocytes decreased the length of STX13-positive recycling endosomal tubules and targeted the SNARE to lysosomes. This result indicates a defect in directing recycling endosomal tubules to melanosomes. Thus, Rab9A and its co-regulatory GTPases control STX13-mediated cargo delivery to maturing melanosomes.

Independent Transport and Sorting of Functionally Distinct Protein Families in Tetrahymena thermophila Dense Core Secretory Granules

Dense core granules (DCGs) in Tetrahymena thermophila contain two protein classes. Proteins in the first class, called granule lattice (Grl), coassemble to form a crystalline lattice within the granule lumen. Lattice expansion acts as a propulsive mechanism during DCG release, and Grl proteins are essential for efficient exocytosis. The second protein class, defined by a C-terminal beta/gamma-crystallin domain, is poorly understood. Here, we have analyzed the function and sorting of Grt1p (granule tip), which was previously identified as an abundant protein in this family. Cells lacking all copies of GRT1, together with the closely related GRT2, accumulate wild-type levels of docked DCGs. Unlike cells disrupted in any of the major GRL genes, Delta GRT1 Delta GRT2 cells show no defect in secretion, indicating that neither exocytic fusion nor core expansion depends on GRT1. These results suggest that Grl protein sorting to DCGs is independent of Grt proteins. Consistent with this, the granule core lattice in Delta GRT1 Delta GRT2 cells appears identical to that in wild-type cells by electron microscopy, and the only biochemical component visibly absent is Grt1p itself. Moreover, gel filtration showed that Grl and Grt proteins in cell homogenates exist in nonoverlapping complexes, and affinity-isolated Grt1p complexes do not contain Grl proteins. These data demonstrate that two major classes of proteins in Tetrahymena DCGs are likely to be independently transported during DCG biosynthesis and play distinct roles in granule function. The role of Grt1p may primarily be postexocytic; consistent with this idea, DCG contents from Delta GRT1 Delta GRT2 cells appear less adhesive than those from the wild type.

Mitochondrial outer membrane proteome of Trypanosoma brucei reveals macromolecular transport machineries and novel factors required to maintain mitochondrial morphology

The mitochondrial outer membrane (MOM) separates the mitochondria from the cytoplasm, serving both as a barrier and as a gateway. Protein complexes — believed to be universally conserved in all eukaryotes — reside in the MOM to orchestrate and control metabolite exchange, lipid metabolism and uptake of biopolymers such as protein and RNA. African trypanosomes are the causative agent of the sleeping sickness in humans. The parasites are among the earliest diverging eukaryotes that have bona fide mitochondria capable of oxidative phosphorylation. Trypanosomes have unique mitochondrial biology that concerns their mitochondrial metabolism and their unusual mitochondrial morphology that differs to great extent between life stages. Another striking feature is the organization of the mitochondrial genome that does not encode any tRNA genes, thus all tRNAs needed for mitochondrial translation have to be imported. However, the MOM of T. brucei is essentially unchartered territory. It lacks a canonical protein import machinery and facilitation of tRNA translocation remains completely elusive. Using biochemical fractionation and label-free quantitative mass spectrometry for correlated protein abundance-profiling we were able to identify a cluster of 82 candidate proteins that can be localized to the trypanosomal MOM with high confidence. This enabled us to identify a highly unusual, potentially archaic protein import machinery that might also transport tRNAs. Moreover, two-thirds of the identified polypeptides present on the MOM have never been associated with mitochondria before. 40 proteins share homology with proteins of known functions. The function of 42 proteins remains unknown. 11 proteins are essential for the disease-causing bloodstream form of T. brucei and therefore may be exploited as novel drug targets. A comparison with the outer membrane proteome of yeast defines a set of 17 common proteins that are likely present in the MOM of all eukaryotes. Known factors involved in the regulation of mitochondrial morphology are virtually absent in T. brucei. Interestingly, RNAi-mediated ablation of three outer membrane proteins of unknown function resulted in a collapse of the network-like mitochondrion of insect-stage parasites and therefore directly or indirectly are involved in the regulation of mitochondrial morphology.

The medial-Golgi Ion Pump Pmr1 Supplies the Yeast Secretory Pathway with Ca2+ and Mn2+ Required for Glycosylation, Sorting, and Endoplasmic Reticulum-Associated Protein Degradation

The yeast Ca2+ adenosine triphosphatase Pmr1, located in medial-Golgi, has been implicated in intracellular transport of Ca2+ and Mn2+ ions. We show here that addition of Mn2+ greatly alleviates defects of pmr1 mutants in N-linked and O-linked protein glycosylation. In contrast, accurate sorting of carboxypeptidase Y (CpY) to the vacuole requires a sufficient supply of intralumenal Ca2+. Most remarkably, pmr1 mutants are also unable to degrade CpY*, a misfolded soluble endoplasmic reticulum protein, and display phenotypes similar to mutants defective in the stress response to malfolded endoplasmic reticulum proteins. Growth inhibition of pmr1 mutants on Ca2+-deficient media is overcome by expression of other Ca2+ pumps, including a SERCA-type Ca2+ adenosine triphosphatase from rabbit, or by Vps10, a sorting receptor guiding non-native luminal proteins to the vacuole. Our analysis corroborates the dual function of Pmr1 in Ca2+ and Mn2+ transport and establishes a novel role of this secretory pathway pump in endoplasmic reticulum-associated processes.

Early Endosomes Are Required for Major Histocompatiblity Complex Class II Transport to Peptide-loading Compartments

Antigen presentation to CD4+ T lymphocytes requires transport of newly synthesized major histocompatibility complex (MHC) class II molecules to the endocytic pathway, where peptide loading occurs. This step is mediated by a signal located in the cytoplasmic tail of the MHC class II-associated Ii chain, which directs the MHC class II-Ii complexes from the trans-Golgi network (TGN) to endosomes. The subcellular machinery responsible for the specific targeting of MHC class II molecules to the endocytic pathway, as well as the first compartments these molecules enter after exit from the TGN, remain unclear. We have designed an original experimental approach to selectively analyze this step of MHC class II transport. Newly synthesized MHC class II molecules were caused to accumulate in the Golgi apparatus and TGN by incubating the cells at 19°C, and early endosomes were functionally inactivated by in vivo cross-linking of transferrin (Tf) receptor–containing endosomes using Tf-HRP complexes and the HRP-insoluble substrate diaminobenzidine. Inactivation of Tf-containing endosomes caused a marked delay in Ii chain degradation, peptide loading, and MHC class II transport to the cell surface. Thus, early endosomes appear to be required for delivery of MHC class II molecules to the endocytic pathway. Under cross-linking conditions, most αβIi complexes accumulated in tubules and vesicles devoid of γ-adaptin and/or mannose-6-phosphate receptor, suggesting an AP1-independent pathway for the delivery of newly synthesized MHC class II molecules from the TGN to endosomes.

Hydrolysis of GTP on rab11 is required for the direct delivery of transferrin from the pericentriolar recycling compartment to the cell surface but not from sorting endosomes

Rab11 is a small GTP-binding protein that in cultured mammalian cells has been shown to be concentrated in the pericentriolar endosomal recycling compartment and to play a key role in passage of the recycling transferrin receptor through that compartment [Ullrich, O., Reinsch, S., Urbé, S., Zerial, M. & Parton, R. G. (1996) J. Cell Biol. 135, 913–924]. To obtain insights into the site(s) of action of rab11 within the recycling pathway, we have now compared the effects on recycling at 37°C of overexpression of wild-type rab11 and various mutant forms of this protein in cells that had been loaded with transferrin at either 37°C or 16°C. We show that incubation at 16°C blocks passage of endocytosed transferrin into the recycling compartment and that, whereas the rab11 dominant negative mutant form (S25N) inhibits transferrin recycling after interiorization at either temperature, the wild-type rab11 and constitutively active mutant (Q70L) have no inhibitory effect on the recycling of molecules that were interiorized at 16°C. This differential inhibitory effect shows that two distinct pathways for recycling are followed by the bulk of the transferrin molecules interiorized at the two different temperatures. The incapacity of the constitutively active form of rab11 (Q70L) to inhibit recycling of molecules interiorized at 16°C is consistent with their recycling taking place directly from sorting endosomes, in a process that does not require hydrolysis of GTP on rab11. The fact that the dominant negative (S25N) form of rab11 inhibits recycling of molecules interiorized at both temperatures indicates that activation of rab11 by GTP is required for exit of transferrin from sorting endosomes, regardless of whether this exit is toward the recycling compartment or directly to the plasma membrane.