Rab-GDI complex dissociation factor expressed through translational frameshifting in filamentous ascomycetes.
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ABSTRACT:
SUBMITTER: Malagnac F
PROVIDER: S-EPMC3777964 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:In eukaryotic cells Rab/Ypt GTPases represent a family of key membrane traffic controllers that associate with their targeted membranes via C-terminally conjugated geranylgeranyl groups. GDP dissociation inhibitor (GDI) is a general and essential regulator of Rab recycling that extracts prenylated Rab proteins from membranes at the end of their cycle of activity and facilitates their delivery to the donor membranes. Here, we present the structure of a complex between GDI and a doubly prenylated Rab protein. We show that one geranylgeranyl residue is deeply buried in a hydrophobic pocket formed by domain II of GDI, whereas the other lipid is more exposed to solvent and is skewed across several atoms of the first moiety. Based on structural information and biophysical measurements, we propose mechanistic and thermodynamic models for GDI and Rab escort protein-mediated interaction of RabGTPase with intracellular membranes.
| S-EPMC1356364 | biostudies-literature
Project description:BACKGROUND: The symptoms of numerous diseases result from genetic mutations that disrupt the homeostasis maintained by the appropriate integration of signaling gene activities. The relationships between signaling genes suggest avenues through which homeostasis can be restored and disease symptoms subsequently reduced. Specifically, disease symptoms caused by loss-of-function mutations in a particular gene may be reduced by concomitant perturbations in genes with antagonistic activities. METHODOLOGY/PRINCIPAL FINDINGS: Here we use network-neighborhood analyses to predict genetic interactions in Caenorhabditis elegans towards mapping antagonisms and synergisms between genes in an animal model. Most of the predicted interactions are novel, and the experimental validation establishes that our approach provides a gain in accuracy compared to previous efforts. In particular, we identified genetic interactors of gdi-1, the orthologue of GDI1, a gene associated with mental retardation in human. Interestingly, some gdi-1 interactors have human orthologues with known neurological functions, and upon validation of the interactions in mammalian systems, these orthologues would be potential therapeutic targets for GDI1-associated neurological disorders. We also observed the conservation of a gdi-1 interaction between different cellular systems in C. elegans, suggesting the involvement of GDI1 in human muscle degeneration. CONCLUSIONS/SIGNIFICANCE: We developed a novel predictor of genetic interactions that may have the ability to significantly streamline the identification of therapeutic targets for monogenic disorders involving genes conserved between human and C. elegans.
| S-EPMC2869356 | biostudies-literature
Project description:The prion protein (PrP) is a glycosylphosphatidylinositol-anchored membrane glycoprotein that plays a vital role in prion diseases, a class of fatal neurodegenerative disorders of humans and animals. Approximately 20% of human prion diseases display autosomal dominant inheritance and are linked to mutations in the PrP gene on chromosome 20. PrP mutations are thought to favor the conformational conversion of PrP into a misfolded isoform that causes disease by an unknown mechanism. The PrP mutation D178N/Met-129 is linked to fatal familial insomnia, which causes severe sleep abnormalities and autonomic dysfunction. We showed by immunoelectron microscopy that this mutant PrP accumulates abnormally in the endoplasmic reticulum and Golgi of transfected neuroblastoma N2a cells. To investigate the impact of intracellular PrP accumulation on cellular homeostasis, we did a two-dimensional gel-based differential proteomics analysis. We used wide range immobilized pH gradient strips, pH 4-7 and 6-11, to analyze a large number of proteins. We found changes in proteins involved in energy metabolism, redox regulation, and vesicular transport. Rab GDP dissociation inhibitor alpha (GDI) was one of the proteins that changed most. GDI regulates vesicular protein trafficking by acting on the activity of several Rab proteins. We found a specific reduction in the level of functional Rab11 in mutant PrP-expressing cells associated with impaired post-Golgi trafficking. Our data are consistent with a model by which mutant PrP induces overexpression of GDI, activating a cytotoxic feedback loop that leads to protein accumulation in the secretory pathway.
| S-EPMC2860234 | biostudies-literature
Project description:The standard rules of genetic translational decoding are altered in specific genes by different events that are globally termed recoding. In Archaea recoding has been unequivocally determined so far only for termination codon readthrough events. We study here the mechanism of expression of a gene encoding for a alpha-l-fucosidase from the archaeon Sulfolobus solfataricus (fucA1), which is split in two open reading frames separated by a -1 frameshifting. The expression in Escherichia coli of the wild-type split gene led to the production by frameshifting of full-length polypeptides with an efficiency of 5%. Mutations in the regulatory site where the shift takes place demonstrate that the expression in vivo occurs in a programmed way. Further, we identify a full-length product of fucA1 in S.solfataricus extracts, which translate this gene in vitro by following programmed -1 frameshifting. This is the first experimental demonstration that this kind of recoding is present in Archaea.
| S-EPMC1616953 | biostudies-literature
Project description:Rab proteins are the major regulators of vesicular trafficking in eukaryotic cells. Their activity can be tightly controlled within cells: Regulated by guanine nucleotide exchange factors (GEFs) and GTPase activating proteins (GAPs), they switch between an active GTP-bound state and an inactive GDP-bound state, interacting with downstream effector proteins only in the active state. Additionally, they can bind to membranes via C-terminal prenylated cysteine residues and they can be solubilized and shuttled between membranes by chaperone-like molecules called GDP dissociation inhibitors (GDIs). In this review we give an overview of Rab proteins with a focus on the current understanding of their regulation by GEFs, GAPs and GDI.
| S-EPMC5902221 | biostudies-literature
Project description:Regulated exocytosis of neurotransmitter from synaptic vesicles involves the function of a small GTP-binding protein, Rab3A. Rab-GDP dissociation inhibitor (GDI) is an important modulator of Rab function and subcellular distribution. We have characterized the respective roles of innervation and target tissue interactions in regulating GDI expression during synapse formation in chick ciliary ganglion (CG) neurons developing in situ. Here we report the first full-length chick GDI cDNA sequence. It is highly homologous to mammalian GDI isoforms and includes all of the sequence-conserved regions critical for Rab3A binding. This chick GDI mRNA is predominantly expressed in neurons as judged by Northern blot analysis of tissue distribution and by in situ hybridization of CG sections. Developmental increases in CG GDI mRNA levels occur in two phases as determined by reverse transcription (RT)-PCR and by Northern analysis of both normal-developing and input- or target tissue-deprived ganglia. The initial phase appears to be independent of cell-cell interactions. In contrast, the second, larger increase is induced by both presynaptic inputs and postganglionic target tissues but does not occur until target tissue innervation. Synaptic interaction with the target seems necessary for the regulatory response to both inputs and target tissues. GDI protein levels show similar changes. The developmentally delayed ability of inputs and targets to influence GDI levels differs from the regulation of neurotransmitter receptor expression in CG neurons. These results suggest that distinct extrinsic regulatory signals influence the expression of synapse-related components at the presynaptic axon terminal versus postsynaptic membrane in an individual neuron.
| S-EPMC6793200 | biostudies-literature
Project description:Photoreceptor outer segments (OS) in the vertebrate retina undergo a process of continual renewal involving shedding of disc membranes that are cleared by phagocytic uptake into the retinal pigment epithelium (RPE). In dystrophic Royal College of Surgeons (RCS) rats, OS phagocytosis is blocked by a mutation in the gene encoding the receptor tyrosine kinase MERTK. To identify proteins tyrosine-phosphorylated downstream of MERTK in the RPE, MALDI-mass spectrometry with peptide-mass fingerprinting was used in comparative studies of RCS congenic and dystrophic rats. At times corresponding to peak phagocytic activity, the RAB GTPase effector GDP dissociation inhibitor alpha (GDI1) was found to undergo tyrosine phosphorylation only in congenic rats. In cryosections of native RPE/choroid, GDI1 colocalized with MERTK and the intracellular tyrosine-kinase SRC. In cultured RPE-J cells, and in transfected heterologous cells, MERTK stimulated SRC-mediated tyrosine phosphorylation of GDI1. In OS-fed RPE-J cells, GDI1 colocalized with MERTK and SRC on apparent phagosomes located near the apical membrane. In addition, both GDI1 and RAB5, a regulator of vesicular transport, colocalized with ingested OS. Taken together, these findings identify a novel role of MERTK signaling in membrane trafficking in the RPE that is likely to subserve mechanisms of phagosome formation.
| S-EPMC4624558 | biostudies-literature
Project description:The nuclear ribosomal DNA (rDNA) is considered as a paradigm of concerted evolution. Components of the rDNA tandem repeats (45S) are widely used in phylogenetic studies of different organisms and the internal transcribed spacer (ITS) region was recently selected as a fungal DNA bar code. However, rRNA pseudogenes, as one kind of escape from concerted evolution, were reported in a wide range of organisms, especially in plants and animals. Moreover, large numbers of 5S rRNA pseudogenes were identified in several filamentous ascomycetes. To study whether rDNA evolves in a strict concerted manner and test whether rRNA pseudogenes exist in more species of ascomycetes, intragenomic rDNA polymorphisms were analyzed using whole genome sequences. Divergent rDNA paralogs were found to coexist within a single genome in seven filamentous ascomycetes examined. A great number of paralogs were identified as pseudogenes according to the mutation and secondary structure analyses. Phylogenetic analyses of the three rRNA coding regions of the 45S rDNA repeats, i.e., 18S, 5.8S, and 28S, revealed an interspecies clustering pattern of those different rDNA paralogs. The identified rRNA pseudogenic sequences were validated using specific primers designed. Mutation analyses revealed that the repeat-induced point (RIP) mutation was probably responsible for the formation of those rRNA pseudogenes.
| S-EPMC5555474 | biostudies-literature
Project description:The Ras-related small GTP-binding proteins are involved in diverse cellular events, including cell signaling, proliferation, cytoskeletal organization, and secretion. The interconversion of the active, GTP-bound form of the protein to the inactive, GDP-bound form is influenced by two types of regulatory proteins, those that alter the intrinsic GTPase activity of the GTP-binding protein and those that affect the rate of GDP/GTP exchange. By utilizing a subtractive hybridization approach, we have isolated a human gene encoding Ly-GDI, a protein that has striking homology to the product of a previously cloned gene, Rho-GDI, which inhibits GDP/GTP exchange on the Rho family of GTPases. In contrast to Rho-GDI, which is ubiquitously expressed, Ly-GDI is expressed only in hematopoietic tissues and predominantly in B- and T-lymphocyte cell lines. The full-length Ly-GDI cDNA encodes a 27-kDa protein which binds to RhoA and inhibits GDP dissociation from RhoA. Stimulation of T lymphocytes with phorbol ester leads to phosphorylation of Ly-GDI, suggesting an involvement of Ly-GDI in lymphocyte activation pathways. Cell type-specific regulators of the Ras-like GTP-binding proteins may provide one mechanism by which different cell types respond uniquely to signals transduced through the same cell surface receptor or may provide a way by which the GTP-binding proteins can be uniquely engaged by tissue-restricted receptors.
| S-EPMC47183 | biostudies-other
Project description:BACKGROUND: Genes responsible for biosynthesis of fungal secondary metabolites are usually tightly clustered in the genome and co-regulated with metabolite production. Epipolythiodioxopiperazines (ETPs) are a class of secondary metabolite toxins produced by disparate ascomycete fungi and implicated in several animal and plant diseases. Gene clusters responsible for their production have previously been defined in only two fungi. Fungal genome sequence data have been surveyed for the presence of putative ETP clusters and cluster data have been generated from several fungal taxa where genome sequences are not available. Phylogenetic analysis of cluster genes has been used to investigate the assembly and heredity of these gene clusters. RESULTS: Putative ETP gene clusters are present in 14 ascomycete taxa, but absent in numerous other ascomycetes examined. These clusters are discontinuously distributed in ascomycete lineages. Gene content is not absolutely fixed, however, common genes are identified and phylogenies of six of these are separately inferred. In each phylogeny almost all cluster genes form monophyletic clades with non-cluster fungal paralogues being the nearest outgroups. This relatedness of cluster genes suggests that a progenitor ETP gene cluster assembled within an ancestral taxon. Within each of the cluster clades, the cluster genes group together in consistent subclades, however, these relationships do not always reflect the phylogeny of ascomycetes. Micro-synteny of several of the genes within the clusters provides further support for these subclades. CONCLUSION: ETP gene clusters appear to have a single origin and have been inherited relatively intact rather than assembling independently in the different ascomycete lineages. This progenitor cluster has given rise to a small number of distinct phylogenetic classes of clusters that are represented in a discontinuous pattern throughout ascomycetes. The disjunct heredity of these clusters is discussed with consideration to multiple instances of independent cluster loss and lateral transfer of gene clusters between lineages.
| S-EPMC2045112 | biostudies-literature