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1.
Cell Mol Life Sci ; 76(20): 4117-4130, 2019 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-31028425

RESUMEN

Intracellular traffic amongst organelles represents a key feature for eukaryotes and is orchestrated principally by members of Rab family, the largest within Ras superfamily. Given that variations in Rab repertoire have been fundamental in animal diversification, we provided the most exhaustive survey regarding the Rab toolkit of chordates. Our findings reveal the existence of 42 metazoan conserved subfamilies exhibiting a univocal intron/exon structure preserved from cnidarians to vertebrates. Since the current view does not capture the Rab complexity, we propose a new Rab family classification in three distinct monophyletic clades. The Rab complement of chordates shows a dramatic diversification due to genome duplications and independent gene duplications and losses with sharp differences amongst cephalochordates, tunicates and gnathostome vertebrates. Strikingly, the analysis of the domain architecture of this family highlighted the existence of chimeric calcium-binding Rabs, which are animal novelties characterized by a complex evolutionary history in gnathostomes and whose role in cellular metabolism is obscure. This work provides novel insights in the knowledge of Rab family: our hypothesis is that chordates represent a hotspot of Rab variability, with many events of gene gains and losses impacting intracellular traffic capabilities. Our results help to elucidate the role of Rab members in the transport amongst endomembranes and shed light on intracellular traffic routes in vertebrates. Then, since the predominant role of Rabs in the molecular communication between different cellular districts, this study paves to way to comprehend inherited or acquired human disorders provoked by dysfunctions in Rab genes.


Asunto(s)
Evolución Biológica , Cordados/genética , Genoma , Familia de Multigenes , Filogenia , Proteínas de Unión al GTP rab/genética , Animales , Transporte Biológico , Cordados/clasificación , Bases de Datos Genéticas , Exones , Duplicación de Gen , Variación Genética , Humanos , Intrones , Orgánulos/genética , Orgánulos/metabolismo , Dominios Proteicos , Sintenía , Proteínas de Unión al GTP rab/clasificación , Proteínas de Unión al GTP rab/metabolismo
2.
Bioinformatics ; 33(4): 568-570, 2017 02 15.
Artículo en Inglés | MEDLINE | ID: mdl-27797763

RESUMEN

Summary: The Rab family of small GTPases regulates and provides specificity to the endomembrane trafficking system; each Rab subfamily is associated with specific pathways. Thus, characterization of Rab repertoires provides functional information about organisms and evolution of the eukaryotic cell. Yet, the complex structure of the Rab family limits the application of existing methods for protein classification. Here, we present a major redesign of the Rabifier, a bioinformatic pipeline for detection and classification of Rab GTPases. It is more accurate, significantly faster than the original version and is now open source, both the code and the data, allowing for community participation. Availability and Implementation: Rabifier and RabDB are freely available through the web at http://rabdb.org . The Rabifier package can be downloaded from the Python Package Index at https://pypi.python.org/pypi/rabifier , the source code is available at Github https://github.com/evocell/rabifier . Contact: jsurkont@igc.gulbenkian.pt or jleal@igc.gulbenkian.pt. Supplementary information: Supplementary data are available at Bioinformatics online.


Asunto(s)
Biología Computacional/métodos , Programas Informáticos , Proteínas de Unión al GTP rab/genética , Animales , Eucariontes/enzimología , Humanos , Proteínas de Unión al GTP rab/clasificación
3.
Planta ; 237(1): 161-72, 2013 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-23001196

RESUMEN

We characterized the gene expression, subcellular localization, and in vivo functions of a Nicotiana benthamiana small GTPase belonging to the RabE family, designated NbRabE1. The NbRabE1 promoter drove strong ß-glucuronidase reporter expression in young tissues containing actively dividing cells and in stomata guard cells. GFP fusion proteins of NbRabE1 and its dominant-negative and constitutively active mutants were all localized to the Golgi apparatus and the plasma membrane but showed different affinities for membrane attachment. Virus-induced gene silencing of NbRabE1 resulted in pleiotropic phenotypes, including growth arrest, premature senescence, and abnormal leaf development. At the cellular level, the leaves in which NbRabE1 was silenced contained abnormal stomata that lacked pores or contained incomplete ventral walls, suggesting that NbRabE1 deficiency leads to defective guard cell cytokinesis. Ectopic expression of the dominant-negative mutant of NbRabE1 in Arabidopsis thaliana resulted in retardation of shoot and root growth accompanied by defective root hair formation. These developmental defects are discussed in conjunction with proposed functions of RabE GTPases in polarized secretory vesicle trafficking.


Asunto(s)
Mutación , Nicotiana/genética , Proteínas de Plantas/genética , Proteínas de Unión al GTP rab/genética , Arabidopsis/genética , Arabidopsis/metabolismo , Western Blotting , Membrana Celular/metabolismo , Regulación de la Expresión Génica de las Plantas , Silenciador del Gen , Glucuronidasa/genética , Glucuronidasa/metabolismo , Aparato de Golgi/metabolismo , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Microscopía Confocal , Microscopía Electrónica de Transmisión , Datos de Secuencia Molecular , Filogenia , Hojas de la Planta/genética , Hojas de la Planta/crecimiento & desarrollo , Proteínas de Plantas/metabolismo , Estomas de Plantas/genética , Estomas de Plantas/crecimiento & desarrollo , Estomas de Plantas/ultraestructura , Virus de Plantas/genética , Plantas Modificadas Genéticamente , Regiones Promotoras Genéticas/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Nicotiana/crecimiento & desarrollo , Nicotiana/virología , Proteínas de Unión al GTP rab/clasificación , Proteínas de Unión al GTP rab/metabolismo
4.
PLoS Genet ; 6(10): e1001155, 2010 Oct 14.
Artículo en Inglés | MEDLINE | ID: mdl-20976245

RESUMEN

Cellular sophistication is not exclusive to multicellular organisms, and unicellular eukaryotes can resemble differentiated animal cells in their complex network of membrane-bound structures. These comparisons can be illuminated by genome-wide surveys of key gene families. We report a systematic analysis of Rabs in a complex unicellular Ciliate, including gene prediction and phylogenetic clustering, expression profiling based on public data, and Green Fluorescent Protein (GFP) tagging. Rabs are monomeric GTPases that regulate membrane traffic. Because Rabs act as compartment-specific determinants, the number of Rabs in an organism reflects intracellular complexity. The Tetrahymena Rab family is similar in size to that in humans and includes both expansions in conserved Rab clades as well as many divergent Rabs. Importantly, more than 90% of Rabs are expressed concurrently in growing cells, while only a small subset appears specialized for other conditions. By localizing most Rabs in living cells, we could assign the majority to specific compartments. These results validated most phylogenetic assignments, but also indicated that some sequence-conserved Rabs were co-opted for novel functions. Our survey uncovered a rare example of a nuclear Rab and substantiated the existence of a previously unrecognized core Rab clade in eukaryotes. Strikingly, several functionally conserved pathways or structures were found to be associated entirely with divergent Rabs. These pathways may have permitted rapid evolution of the associated Rabs or may have arisen independently in diverse lineages and then converged. Thus, characterizing entire gene families can provide insight into the evolutionary flexibility of fundamental cellular pathways.


Asunto(s)
Membrana Celular/metabolismo , Proteínas Protozoarias/genética , Tetrahymena thermophila/genética , Proteínas de Unión al GTP rab/genética , Animales , Transporte Biológico , Western Blotting , Endocitosis , Células Eucariotas/metabolismo , Evolución Molecular , Genoma de Protozoos , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Microscopía Fluorescente , Familia de Multigenes , Fagocitosis , Filogenia , Proteínas Protozoarias/metabolismo , Transducción de Señal , Tetrahymena thermophila/metabolismo , Proteínas de Unión al GTP rab/clasificación , Proteínas de Unión al GTP rab/metabolismo
5.
BMC Biol ; 10: 71, 2012 Aug 08.
Artículo en Inglés | MEDLINE | ID: mdl-22873208

RESUMEN

BACKGROUND: Membrane-bound organelles are a defining feature of eukaryotic cells, and play a central role in most of their fundamental processes. The Rab G proteins are the single largest family of proteins that participate in the traffic between organelles, with 66 Rabs encoded in the human genome. Rabs direct the organelle-specific recruitment of vesicle tethering factors, motor proteins, and regulators of membrane traffic. Each organelle or vesicle class is typically associated with one or more Rab, with the Rabs present in a particular cell reflecting that cell's complement of organelles and trafficking routes. RESULTS: Through iterative use of hidden Markov models and tree building, we classified Rabs across the eukaryotic kingdom to provide the most comprehensive view of Rab evolution obtained to date. A strikingly large repertoire of at least 20 Rabs appears to have been present in the last eukaryotic common ancestor (LECA), consistent with the 'complexity early' view of eukaryotic evolution. We were able to place these Rabs into six supergroups, giving a deep view into eukaryotic prehistory. CONCLUSIONS: Tracing the fate of the LECA Rabs revealed extensive losses with many extant eukaryotes having fewer Rabs, and none having the full complement. We found that other Rabs have expanded and diversified, including a large expansion at the dawn of metazoans, which could be followed to provide an account of the evolutionary history of all human Rabs. Some Rab changes could be correlated with differences in cellular organization, and the relative lack of variation in other families of membrane-traffic proteins suggests that it is the changes in Rabs that primarily underlies the variation in organelles between species and cell types.


Asunto(s)
Evolución Molecular , Genómica , Proteínas de Unión al GTP rab/genética , Secuencia de Aminoácidos , Animales , Eucariontes/genética , Variación Genética , Humanos , Cadenas de Markov , Familia de Multigenes , Filogenia , Reproducibilidad de los Resultados , Especificidad de la Especie , Proteínas de Unión al GTP rab/química , Proteínas de Unión al GTP rab/clasificación
6.
PLoS Comput Biol ; 7(10): e1002217, 2011 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-22022256

RESUMEN

Rab proteins are small GTPases that act as essential regulators of vesicular trafficking. 44 subfamilies are known in humans, performing specific sets of functions at distinct subcellular localisations and tissues. Rab function is conserved even amongst distant orthologs. Hence, the annotation of Rabs yields functional predictions about the cell biology of trafficking. So far, annotating Rabs has been a laborious manual task not feasible for current and future genomic output of deep sequencing technologies. We developed, validated and benchmarked the Rabifier, an automated bioinformatic pipeline for the identification and classification of Rabs, which achieves up to 90% classification accuracy. We cataloged roughly 8.000 Rabs from 247 genomes covering the entire eukaryotic tree. The full Rab database and a web tool implementing the pipeline are publicly available at www.RabDB.org. For the first time, we describe and analyse the evolution of Rabs in a dataset covering the whole eukaryotic phylogeny. We found a highly dynamic family undergoing frequent taxon-specific expansions and losses. We dated the origin of human subfamilies using phylogenetic profiling, which enlarged the Rab repertoire of the Last Eukaryotic Common Ancestor with Rab14, 32 and RabL4. Furthermore, a detailed analysis of the Choanoflagellate Monosiga brevicollis Rab family pinpointed the changes that accompanied the emergence of Metazoan multicellularity, mainly an important expansion and specialisation of the secretory pathway. Lastly, we experimentally establish tissue specificity in expression of mouse Rabs and show that neo-functionalisation best explains the emergence of new human Rab subfamilies. With the Rabifier and RabDB, we provide tools that easily allows non-bioinformaticians to integrate thousands of Rabs in their analyses. RabDB is designed to enable the cell biology community to keep pace with the increasing number of fully-sequenced genomes and change the scale at which we perform comparative analysis in cell biology.


Asunto(s)
Proteínas de Unión al GTP rab/metabolismo , Animales , Bases de Datos de Proteínas , Humanos , Filogenia , Transporte de Proteínas , Proteínas de Unión al GTP rab/clasificación
7.
Traffic ; 10(7): 912-24, 2009 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-19453973

RESUMEN

Small guanine triphosphatases (GTPases) of the Rab family are key regulators of membrane trafficking events between the various subcellular compartments in eukaryotic cells. Rab7 is a conserved protein required in the late endocytic pathway and in lysosome biogenesis. A Schizosaccharomyces pombe (S. pombe) homolog of Rab7, Ypt7, is necessary for trafficking from the endosome to the vacuole and for homotypic vacuole fusion. Here, we identified and characterized a second fission yeast Rab7 homolog, Ypt71. Ypt71 is localized to the vacuolar membrane. Cells deleted for ypt71(+) exhibit normal growth rates and morphology. Interestingly, a ypt71 null mutant contains large vacuoles in contrast with the small fragmented vacuoles found in the ypt7 null mutant. Furthermore, the ypt71 mutation does not enhance or alleviate the temperature sensitivity or vacuole fusion defect of ypt7Delta cells. Like ypt7Delta cells, overexpression of ypt71(+) caused fragmentation of vacuoles and inhibits vacuole fusion under hypotonic conditions. Thus, the two S. pombe Rab7 homologs act antagonistically in regulating vacuolar morphology. Analysis of a chimeric Ypt7/Ypt71 protein showed that Rab7-directed vacuole dynamics, fusion versus fission, largely depends on the medial region of the protein, including a part of RabSF3/alpha3-L7.


Asunto(s)
Isoformas de Proteínas/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces , Vacuolas/ultraestructura , Proteínas de Unión al GTP rab/metabolismo , Secuencia de Aminoácidos , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Filogenia , Isoformas de Proteínas/clasificación , Isoformas de Proteínas/genética , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Schizosaccharomyces/citología , Schizosaccharomyces/metabolismo , Proteínas de Schizosaccharomyces pombe/clasificación , Proteínas de Schizosaccharomyces pombe/genética , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Vacuolas/metabolismo , Proteínas de Unión al GTP rab/clasificación , Proteínas de Unión al GTP rab/genética , Proteínas de Unión a GTP rab7
8.
FEBS J ; 288(1): 36-55, 2021 01.
Artículo en Inglés | MEDLINE | ID: mdl-32542850

RESUMEN

The Rab family of small GTPases regulates intracellular membrane trafficking by orchestrating the biogenesis, transport, tethering, and fusion of membrane-bound organelles and vesicles. Like other small GTPases, Rabs cycle between two states, an active (GTP-loaded) state and an inactive (GDP-loaded) state, and their cycling is catalyzed by guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs). Because an active form of each Rab localizes on a specific organelle (or vesicle) and recruits various effector proteins to facilitate each step of membrane trafficking, knowing when and where Rabs are activated and what effectors Rabs recruit is crucial to understand their functions. Since the discovery of Rabs, they have been regarded as one of the central hubs for membrane trafficking, and numerous biochemical and genetic studies have revealed the mechanisms of Rab functions in recent years. The results of these studies have included the identification and characterization of novel GEFs, GAPs, and effectors, as well as post-translational modifications, for example, phosphorylation, of Rabs. Rab functions beyond the simple effector-recruiting model are also emerging. Furthermore, the recently developed CRISPR/Cas technology has enabled acceleration of knockout analyses in both animals and cultured cells and revealed previously unknown physiological roles of many Rabs. In this review article, we provide the most up-to-date and comprehensive lists of GEFs, GAPs, effectors, and knockout phenotypes of mammalian Rabs and discuss recent findings in regard to their regulation and functions.


Asunto(s)
Proteínas Activadoras de GTPasa/genética , Factores de Intercambio de Guanina Nucleótido/genética , Orgánulos/metabolismo , Procesamiento Proteico-Postraduccional , Vesículas Transportadoras/metabolismo , Proteínas de Unión al GTP rab/genética , Animales , Transporte Biológico , Células Eucariotas/citología , Células Eucariotas/metabolismo , Proteínas Activadoras de GTPasa/clasificación , Proteínas Activadoras de GTPasa/metabolismo , Factores de Intercambio de Guanina Nucleótido/clasificación , Factores de Intercambio de Guanina Nucleótido/metabolismo , Guanosina Difosfato/metabolismo , Guanosina Trifosfato/metabolismo , Humanos , Orgánulos/química , Fosforilación , Filogenia , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Terminología como Asunto , Vesículas Transportadoras/química , Proteínas de Unión al GTP rab/clasificación , Proteínas de Unión al GTP rab/metabolismo
9.
Mol Biol (Mosk) ; 44(1): 20-7, 2010.
Artículo en Inglés | MEDLINE | ID: mdl-20198855

RESUMEN

Complete coding sequences of three Black-boned sheep (Ovis aries) genes Rab2A, Rab3A and Rab7A were amplified using reverse transcription polymerase chain reaction (RT-PCR) based on the conserved sequence information of cattle or other mammals known to be highly homologous to sheep ESTs. The Black-boned sheep Rab2A gene encodes a protein of 226 amino acids which contains the conserved putative RabL2 domain and is highly homologous to the Rab2A proteins of seven other species--cattle (96%), human (83%), Sumatran orangutan (82%), rat (81%), mouse (80%), African clawed frog (72%) and zebrafish (71%). The Black-boned sheep Rab3A gene encodes a protein of 220 amino acids that contains the conserved putative Rab3 domain and is very similar to the Rab3A proteins of four species--cattle (99%), African clawed frog (99%), Western clawed frog (98%) and zebrafish (95%). And the Black-boned sheep Rab7A gene encodes a protein of 207 amino acids that contains the conserved putative Rab7 domain and has high homology with the Rab7A proteins of six other species--human (99%), dog (99%), Sumatran orangutan (99%), zebrafish (97%), rabbit (97%) and African clawed frog (96%). Analysis of the phylogenetic tree has demonstrated that the Black-boned sheep Rab2A, Rab3A and Rab7A proteins share a common ancestor and the tissue expression analysis has shown that the corresponding genes are expressed in a range of tissues including leg muscle, kidney, skin, longissimus dorsi muscle, spleen, heart and liver. Our experiment is the first to provide the primary foundation for a further insight into these three sheep genes.


Asunto(s)
Oveja Doméstica/genética , Proteínas de Unión al GTP rab/genética , Proteína de Unión al GTP rab2/genética , Proteína de Unión al GTP rab3A/genética , Secuencia de Aminoácidos , Animales , Bovinos , Perros , Perfilación de la Expresión Génica , Humanos , Ratones , Datos de Secuencia Molecular , Filogenia , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Conejos , Ratas , Distribución Tisular , Proteínas de Unión al GTP rab/química , Proteínas de Unión al GTP rab/clasificación , Proteína de Unión al GTP rab2/química , Proteína de Unión al GTP rab2/clasificación , Proteína de Unión al GTP rab3A/química , Proteína de Unión al GTP rab3A/clasificación , Proteínas de Unión a GTP rab7
10.
Dev Cell ; 7(5): 663-76, 2004 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-15525528

RESUMEN

In many higher organisms, 5%-15% of histone H2A is ubiquitylated at lysine 119 (uH2A). The function of this modification and the factors involved in its establishment, however, are unknown. Here we demonstrate that uH2A occurs on the inactive X chromosome in female mammals and that this correlates with recruitment of Polycomb group (PcG) proteins belonging to Polycomb repressor complex 1 (PRC1). Based on our observations, we tested the role of the PRC1 protein Ring1B and its closely related homolog Ring1A in H2A ubiquitylation. Analysis of Ring1B null embryonic stem (ES) cells revealed extensive depletion of global uH2A levels. On the inactive X chromosome, uH2A was maintained in Ring1A or Ring1B null cells, but not in double knockout cells, demonstrating an overlapping function for these proteins in development. These observations link H2A ubiquitylation, X inactivation, and PRC1 PcG function, suggesting an unanticipated and novel mechanism for chromatin-mediated heritable gene silencing.


Asunto(s)
Proteínas Portadoras/metabolismo , Compensación de Dosificación (Genética) , Silenciador del Gen , Histonas/metabolismo , Ubiquitina/metabolismo , Proteínas de Unión al GTP rab/metabolismo , Animales , Anticuerpos Monoclonales/metabolismo , Blastocisto/metabolismo , Western Blotting , Proteínas Portadoras/clasificación , Proteínas Portadoras/genética , Línea Celular , Cruzamientos Genéticos , Embrión de Mamíferos/citología , Femenino , Fibroblastos/metabolismo , Eliminación de Gen , Marcación de Gen , Histonas/aislamiento & purificación , Péptidos y Proteínas de Señalización Intracelular , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos CBA , Mapeo Restrictivo , Células Madre/metabolismo , Proteínas de Unión al GTP rab/clasificación , Proteínas de Unión al GTP rab/genética
11.
Methods Enzymol ; 403: 19-28, 2005.
Artículo en Inglés | MEDLINE | ID: mdl-16473574

RESUMEN

Researchers looking to solve biological problems have access to enormous amounts of sequence information and the desktop computational infrastructure to personally interrogate and analyze large datasets. Many powerful bioinformatics tools are available online; however, this discourages the customized analysis of data that is necessary for the experimental scientist to make maximally effective use of the information. In addition, a customized environment facilitates the critical evaluation of bioinformatic methods. This chapter presents a protocol developed to aid in classification of subfamilies and subclasses of a superfamily using the personal desktop computer. The visual representation of the qualitative and quantitative results of data analyses is also considered. The examples are focused on Rab GTPases but are more widely applicable to the classification of any given protein family.


Asunto(s)
Algoritmos , Filogenia , Proteínas de Unión al GTP rab/metabolismo , Secuencia de Aminoácidos , Datos de Secuencia Molecular , Homología de Secuencia de Aminoácido , Proteínas de Unión al GTP rab/química , Proteínas de Unión al GTP rab/clasificación
12.
Contraception ; 72(4): 280-90, 2005 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-16181972

RESUMEN

For the past several years, studies from other laboratories, as well as ours, have begun to unravel the mechanism of germ cell movement in the testis by using several in vitro and in vivo models of tight and adherens junction assembly and disassembly, two cellular phenomena that confer cell movement. However, for cell movement to be fully appreciated, the importance of "intracellular" cell movements, such as those involving actin and microtubule filaments, must be better understood. Recent research on Rab GTPases has shown that members of this superfamily function in the trafficking of vesicles containing cargo to distinct subcellular sites such as the plasma membrane while utilizing actin and microtubule filaments as tracks. In this mini-review, we provide an overview of Rab GTPase structure, function, and regulation, while placing added emphasis on the role of Rabs in cell junction dynamics in the testis.


Asunto(s)
Uniones Intercelulares/fisiología , Proteínas de Unión al GTP rab/fisiología , Animales , Transporte Biológico , Citoesqueleto/ultraestructura , Endocitosis , Exocitosis , Homeostasis , Humanos , Masculino , Fosforilación , Prenilación de Proteína , Relación Estructura-Actividad , Testículo/ultraestructura , Proteínas de Unión al GTP rab/análisis , Proteínas de Unión al GTP rab/química , Proteínas de Unión al GTP rab/clasificación
13.
Methods Mol Biol ; 1298: 17-28, 2015.
Artículo en Inglés | MEDLINE | ID: mdl-25800829

RESUMEN

The bioinformatic annotation of Rab GTPases is important, for example, to understand the evolution of the endomembrane system. However, Rabs are particularly challenging for standard annotation pipelines because they are similar to other small GTPases and form a large family with many paralogous subfamilies. Here, we describe a bioinformatic annotation pipeline specifically tailored to Rab GTPases. It proceeds in two steps: first, Rabs are distinguished from other proteins based on GTPase-specific motifs, overall sequence similarity to other Rabs, and the occurrence of Rab-specific motifs. Second, Rabs are classified taking either a more accurate but slower phylogenetic approach or a slightly less accurate but much faster bioinformatic approach. All necessary steps can either be performed locally or using the referenced online tools. An implementation of a slightly more involved version of the pipeline presented here is available at RabDB.org.


Asunto(s)
Biología Computacional/métodos , Proteínas de Unión al GTP rab/clasificación , Secuencia de Aminoácidos , Humanos , Datos de Secuencia Molecular , Proteínas de Unión al GTP rab/química , Proteínas de Unión al GTP rab/metabolismo
14.
DNA Cell Biol ; 34(2): 83-91, 2015 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-25408252

RESUMEN

The endomembrane system plays an important role during cellular adaptation of the plants with the extracellular environment. The small GTP-binding protein Rab7 located at the vacuolar membrane regulates the vesicle fusion with the vacuole and thereby helps in recycling of the molecules. This is the first report on isolation and characterization of AlRab7 gene from the halophyte plant, Aeluropus that extrudes NaCl through salt glands and grows luxuriantly throughout the year at the Gujarat coast, India. The AlRab7 encodes a protein with 206 amino acids, and a highly conserved effector-binding domain and four nucleotide-binding domains. The in silico analysis predicts the presence of the prenylation site for Rab geranylgeranyltransferase 2 and the Rab escort protein site. The C-terminal two cysteine residues in -XCC sequence are present for membrane attachment. Transcript expression of the AlRab7 gene was differentially regulated by different environmental stimuli such as dehydration, salinity, and hormone abscisic acid (ABA). The recombinant Escherichia coli cells showed improved growth in Luria Bertani medium supplemented with NaCl, KCl, mannitol, ABA, and indole-3-acetic acid. A novel Rab7 interacting partner AlRabring7 was identified by yeast two-hybrid screening.


Asunto(s)
Proteínas de Plantas/genética , Poaceae/genética , Plantas Tolerantes a la Sal/genética , Proteínas de Unión al GTP rab/genética , Ácido Abscísico/farmacología , Secuencia de Aminoácidos , Secuencia de Bases , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Clonación Molecular , ADN Complementario/química , ADN Complementario/genética , Regulación de la Expresión Génica de las Plantas/efectos de los fármacos , Datos de Secuencia Molecular , Filogenia , Reguladores del Crecimiento de las Plantas/farmacología , Proteínas de Plantas/metabolismo , Poaceae/metabolismo , Unión Proteica , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Plantas Tolerantes a la Sal/metabolismo , Análisis de Secuencia de ADN , Homología de Secuencia de Aminoácido , Homología de Secuencia de Ácido Nucleico , Cloruro de Sodio/metabolismo , Cloruro de Sodio/farmacología , Vesículas Transportadoras/metabolismo , Técnicas del Sistema de Dos Híbridos , Agua/farmacología , Proteínas de Unión al GTP rab/clasificación , Proteínas de Unión al GTP rab/metabolismo , Proteínas de Unión a GTP rab7
15.
Science ; 338(6109): 960-3, 2012 Nov 16.
Artículo en Inglés | MEDLINE | ID: mdl-23162001

RESUMEN

Unlike other Salmonellae, the intracellular bacterial human pathogen Salmonella Typhi exhibits strict host specificity. The molecular bases for this restriction are unknown. Here we found that the expression of a single type III secretion system effector protein from broad-host Salmonella Typhimurium allowed Salmonella Typhi to survive and replicate within macrophages and tissues from mice, a nonpermissive host. This effector proteolytically targeted Rab32, which controls traffic to lysosome-related organelles in conjunction with components of the biogenesis of lysosome-related organelle complexes (BLOCs). RNA interference-mediated depletion of Rab32 or of an essential component of a BLOC complex was sufficient to allow S. Typhi to survive within mouse macrophages. Furthermore, S. Typhi was able to survive in macrophages from mice defective in BLOC components.


Asunto(s)
Sistemas de Secreción Bacterianos/fisiología , Interacciones Huésped-Patógeno , Salmonella typhi/fisiología , Proteínas de Unión al GTP rab/fisiología , Secuencia de Aminoácidos , Animales , Sistemas de Secreción Bacterianos/genética , Células COS , Chlorocebus aethiops , Humanos , Lisosomas/metabolismo , Ratones , Ratones Endogámicos C57BL , Datos de Secuencia Molecular , Filogenia , Interferencia de ARN , Salmonella typhi/genética , Proteínas de Unión al GTP rab/clasificación , Proteínas de Unión al GTP rab/genética
16.
PLoS One ; 7(11): e49387, 2012.
Artículo en Inglés | MEDLINE | ID: mdl-23185324

RESUMEN

Rab monomeric GTPases regulate specific aspects of vesicle transport in eukaryotes including coat recruitment, uncoating, fission, motility, target selection and fusion. Moreover, individual Rab proteins function at specific sites within the cell, for example the ER, golgi and early endosome. Importantly, the localization and function of individual Rab subfamily members are often conserved underscoring the significant contributions that model organisms such as Caenorhabditis elegans can make towards a better understanding of human disease caused by Rab and vesicle trafficking malfunction. With this in mind, a bioinformatics approach was first taken to identify and classify the complete C. elegans Rab family placing individual Rabs into specific subfamilies based on molecular phylogenetics. For genes that were difficult to classify by sequence similarity alone, we did a comparative analysis of intron position among specific subfamilies from yeast to humans. This two-pronged approach allowed the classification of 30 out of 31 C. elegans Rab proteins identified here including Rab31/Rab50, a likely member of the last eukaryotic common ancestor (LECA). Second, a molecular toolset was created to facilitate research on biological processes that involve Rab proteins. Specifically, we used Gateway-compatible C. elegans ORFeome clones as starting material to create 44 full-length, sequence-verified, dominant-negative (DN) and constitutive active (CA) rab open reading frames (ORFs). Development of this toolset provided independent research projects for students enrolled in a research-based molecular techniques course at California State University, East Bay (CSUEB).


Asunto(s)
Proteínas de Caenorhabditis elegans/clasificación , Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/enzimología , Biología Computacional/métodos , Familia de Multigenes , Proteínas de Unión al GTP rab/clasificación , Proteínas de Unión al GTP rab/metabolismo , Secuencia de Aminoácidos , Animales , Proteínas de Caenorhabditis elegans/química , Células Clonales , Secuencia Conservada/genética , Humanos , Intrones/genética , Datos de Secuencia Molecular , Sistemas de Lectura Abierta/genética , Filogenia , Empalme del ARN/genética , Reproducibilidad de los Resultados , Alineación de Secuencia , Proteínas de Unión al GTP rab/química
17.
Curr Opin Plant Biol ; 11(6): 610-9, 2008 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-18952493

RESUMEN

Rab GTPases are important determinants of membrane identity and membrane targeting. Higher plants have evolved a unique set of Rab GTPases that presumably reflects the specific demands of plant cell trafficking. In recent years, significant progress has been made in identifying Rab GTPases involved in endosome organisation, cytokinesis and in post-Golgi traffic to the plasma membrane and vacuoles. These include members of the Rab-F1, Rab-F2, Rab-A1, Rab-A2 and Rab-A4 subclasses. Some important regulators or effectors have also been identified for Rab-F, Rab-A1 and Rab-A4 proteins. However, uncertainties remain about the trafficking pathways that connect the compartments in the trans-Golgi/prevacuolar/endosomal system and there is still little or no insight into the functions of several major subclasses within the Rab GTPase family.


Asunto(s)
Membrana Celular/enzimología , Células Vegetales , Plantas/enzimología , Proteínas de Unión al GTP rab/metabolismo , Transporte Biológico , Citocinesis , Vacuolas/enzimología , Proteínas de Unión al GTP rab/clasificación
18.
Cell Mol Life Sci ; 65(18): 2801-13, 2008 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-18726178

RESUMEN

Secretion is a fundamental biological activity of all eukaryotic cells by which they release certain substances in the extracellular space. It is considered a specialized mode of membrane trafficking that is achieved by docking and fusion of secretory vesicles to the plasma membrane (i.e., exocytosis). Secretory vesicle traffic is thought to be regulated by a family of Rab small GTPases, which are regulators of membrane traffic that are common to all eukaryotic cells. Classically, mammalian Rab3 subfamily members were thought to be critical regulators of secretory vesicle exocytosis in neurons and endocrine cells, but recent genetic and proteomic studies indicate that Rab3 is not the sole Rab isoform that regulates secretory vesicle traffic. Rather, additional Rab isoforms, especially Rab27 subfamily members, are required for this process. In this article I review the current literature on the function of Rab isoforms and their effectors in regulated secretory vesicle traffic.


Asunto(s)
Exocitosis/fisiología , Isoenzimas/metabolismo , Vesículas Secretoras/metabolismo , Proteínas de Unión al GTP rab/metabolismo , Animales , Transporte Biológico/fisiología , Humanos , Isoenzimas/clasificación , Isoenzimas/genética , Filogenia , Estructura Terciaria de Proteína , Proteínas de Unión al GTP rab/clasificación , Proteínas de Unión al GTP rab/genética
19.
Exp Parasitol ; 110(3): 244-52, 2005 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-15955319

RESUMEN

Rab proteins are ubiquitous small GTP-binding proteins that form a highly conserved family and regulate vesicular trafficking. Recent completion of the genome of the enteric protozoan parasite Entamoeba histolytica enabled us to identify an extremely large number (>90) of putative Rab genes. Multiple alignment and phylogenic analysis of amebic, human, and yeast Rab showed that only 22 amebic Rab proteins including EhRab1, EhRab2, EhRab5, EhRab7, EhRab8, EhRab11, and EhRab21 showed significant similarity to Rab from other organisms. The 69 remaining amebic Rab proteins showed only moderate similarity (<40% identity) to Rab proteins from other organisms. Approximately one-third of Rab proteins including Rab7, Rab11, and RabC form 15 subfamilies, which contain up to nine isoforms. Approximately 70% of amebic Rab genes contain single or multiple introns, and this proportion is significantly higher than that of common genes in this organism. Twenty-five Rabs possess an atypical carboxyl terminus such as CXXX, XCXX, XXCX, XXXC, and no cysteine. We propose annotation of amebic Rab genes and discuss biological significance of this extraordinary diversity of EhRab proteins in this organism.


Asunto(s)
Entamoeba histolytica/enzimología , Proteínas de Unión al GTP rab/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Entamoeba histolytica/clasificación , Entamoeba histolytica/genética , Variación Genética , Humanos , Datos de Secuencia Molecular , Filogenia , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Proteínas de Unión al GTP rab/química , Proteínas de Unión al GTP rab/clasificación
20.
Biochem Biophys Res Commun ; 338(3): 1607-16, 2005 Dec 23.
Artículo en Inglés | MEDLINE | ID: mdl-16288726

RESUMEN

Endosymbiotic association of the Symbiodinium dinoflagellates (zooxanthellae) with their cnidarian host cells involves an alteration in the development of the alga-enclosing phagosomes. To uncover its molecular basis, we previously investigated and established that the intracellular persistence of the zooxanthella-containing phagosomes involves specific alga-mediated interference with the expression of ApRab5 and ApRab7, two key endocytic regulatory Rab proteins, which results in the selective retention of the former on and exclusion of the later from the organelles. Here we examined the role of ApRab11, a cnidarian homologue of the key endocytic recycling regulator, Rab11, in the Aiptasia-Symbiodinium endosymbiosis. ApRab11 protein shared 88% overall sequence identity with human Rab11A and contained all Rab-specific signature motifs. Co-localization and mutagenesis studies showed that EGFP-tagged ApRab11 was predominantly associated with recycling endosomes and functioned in the recycling of internalized transferrin. In phagocytosis of latex beads, ApRab11 was quickly recruited to and later gradually removed from the developing phagosomes. Significantly, although ApRab11 immunoreactivity was rapidly detected on the phagosomes containing either newly internalized, heat-killed zooxanthellae, or resident zooxanthellae briefly treated with the photosynthesis inhibitor DCMU, it was rarely observed in the majority of phagosomes containing either newly internalized live, or healthy resident, zooxanthellae. It was concluded that through active exclusion of ApRab11 from the phagosomes in which they reside, zooxanthellae interfere with the normal recycling process required for efficient phagosome maturation, and thereby, secure their intracellular persistence, and consequently their endosymbiotic relationship with their cnidarian hosts.


Asunto(s)
Antozoos/metabolismo , Antozoos/parasitología , Dinoflagelados/fisiología , Simbiosis , Proteínas de Unión al GTP rab/metabolismo , Secuencia de Aminoácidos , Animales , Antozoos/química , Antozoos/genética , Secuencia de Bases , Clonación Molecular , ADN Complementario/genética , ADN Complementario/metabolismo , Endocitosis , Humanos , Datos de Secuencia Molecular , Fagocitosis , Filogenia , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Factores de Tiempo , Proteínas de Unión al GTP rab/química , Proteínas de Unión al GTP rab/clasificación , Proteínas de Unión al GTP rab/genética
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