Giardia intestinalis is an enteric pathogen with an extremely modified membrane trafficking system, lacking canonical compartments such as the Golgi, endosomes, and intermediate vesicle carriers. By comparison the fornicate relatives of Giardia possess greater endomembrane system complexity. In eukaryotes, the ADP ribosylation factor (ARF) GTPase regulatory system proteins, which consist of the small GTPase ARF1, and its guanine exchange nucleotide factors (GEFs) and GTPase activating proteins (GAPs), coordinate temporal and directional trafficking of cargo vesicles by recognizing and interacting with heterotetrameric coat complexes at pre-Golgi and post-Golgi interfaces. To understand the evolution of this regulatory system across the fornicate lineage, we have performed comparative genomic and phylogenetic analyses of the ARF GTPases, and their regulatory GAPs and GEFs in fornicate genomes and transcriptomes. Prior to our analysis of the fornicates, we first establish that the ARF GAP sub-family ArfGAP with dual PH domains (ADAP) is sparsely distributed but present in at least four eukaryotic supergroups and thus was likely present in the Last Eukaryotic Common Ancestor (LECA). Next, our collective comparative genomic and phylogenetic investigations into the ARF regulatory proteins in fornicates identify a duplication of ARF1 GTPase yielding two paralogues of ARF1F proteins, ancestral to all fornicates and present in all examined isolates of Giardia. However, the ARF GEF and ARF GAP complement is reduced compared with the LECA. This investigation shows that the system was significantly streamlined prior to the fornicate ancestor but was not further reduced concurrent with a transition into a parasitic lifestyle.

• MeSH
• ADP-ribosylační faktory * genetika   metabolismus   MeSH
• fylogeneze MeSH
• Giardia lamblia * genetika   metabolismus   MeSH
• proteiny aktivující GTPasu genetika   metabolismus   MeSH
• výměnné faktory guaninnukleotidů genetika   metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Staufen1 (STAU1) is a dsRNA binding protein mediating mRNA transport and localization, translational control and STAU1-mediated mRNA decay (SMD). The STAU1 binding site (SBS) within human ADP-ribosylation factor1 (ARF1) 3'UTR binds STAU1 and this downregulates ARF1 cytoplasmic mRNA levels by SMD. However, how STAU1 recognizes specific mRNA targets is still under debate. Our structure of the ARF1 SBS-STAU1 complex uncovers target recognition by STAU1. STAU1 dsRNA binding domain (dsRBD) 4 interacts with two pyrimidines and one purine from the minor groove side via helix α1, the β1-β2 loop anchors the dsRBD at the end of the dsRNA and lysines in helix α2 bind to the phosphodiester backbone from the major groove side. STAU1 dsRBD3 displays the same binding mode with specific recognition of one guanine base. Mutants disrupting minor groove recognition of ARF1 SBS affect in vitro binding and reduce SMD in vivo. Our data thus reveal how STAU1 recognizes minor groove features in dsRNA relevant for target selection.

• MeSH
• ADP-ribosylační faktor 1 chemie   genetika   MeSH
• cytoplazma chemie   genetika   MeSH
• cytoskeletální proteiny chemie   genetika   MeSH
• dvouvláknová RNA chemie   genetika   MeSH
• konformace proteinů MeSH
• lidé MeSH
• proteiny vázající RNA chemie   genetika   MeSH
• stabilita RNA genetika   MeSH
• vazebná místa genetika   MeSH
• vazebný motiv pro dvoušroubovici RNA genetika   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Abiotic stress poses constant challenges for plant survival and is a serious problem for global agricultural productivity. On a molecular level, stress conditions result in elevation of reactive oxygen species (ROS) production causing oxidative stress associated with oxidation of proteins and nucleic acids as well as impairment of membrane functions. Adaptation of root growth to ROS accumulation is facilitated through modification of auxin and cytokinin hormone homeostasis. Here, we report that in Arabidopsis root meristem, ROS-induced changes of auxin levels correspond to decreased abundance of PIN auxin efflux carriers at the plasma membrane (PM). Specifically, increase in H2O2 levels affects PIN2 endocytic recycling. We show that the PIN2 intracellular trafficking during adaptation to oxidative stress requires the function of the ADP-ribosylation factor (ARF)-guanine-nucleotide exchange factor (GEF) BEN1, an actin-associated regulator of the trafficking from the PM to early endosomes and, presumably, indirectly, trafficking to the vacuoles. We propose that H2O2 levels affect the actin dynamics thus modulating ARF-GEF-dependent trafficking of PIN2. This mechanism provides a way how root growth acclimates to stress and adapts to a changing environment.

• MeSH
• ADP-ribosylační faktory metabolismus   fyziologie   MeSH
• aktiny metabolismus   MeSH
• alkoholoxidoreduktasy metabolismus   fyziologie   MeSH
• Arabidopsis metabolismus   fyziologie   MeSH
• cytoskelet metabolismus   MeSH
• fyziologická adaptace MeSH
• kořeny rostlin metabolismus   fyziologie   MeSH
• oxidační stres * MeSH
• peroxid vodíku metabolismus   MeSH
• proteiny huseníčku metabolismus   fyziologie   MeSH
• reaktivní formy kyslíku metabolismus   MeSH
• výměnné faktory guaninnukleotidů metabolismus   fyziologie   MeSH
• Publikační typ
• časopisecké články MeSH

Bardet-Biedl syndrome (BBS) is a recessive genetic disease causing multiple organ anomalies. Most patients carry mutations in genes encoding for the subunits of the BBSome, an octameric ciliary transport complex, or accessory proteins involved in the BBSome assembly or function. BBS proteins have been extensively studied using in vitro, cellular, and animal models. However, the molecular functions of particular BBS proteins and the etiology of the BBS symptoms are still largely elusive. In this study, we applied a meta-analysis approach to study the genotype-phenotype association in humans using our database of all reported BBS patients. The analysis revealed that the identity of the causative gene and the character of the mutation partially predict the clinical outcome of the disease. Besides their potential use for clinical prognosis, our analysis revealed functional differences of particular BBS genes in humans. Core BBSome subunits BBS2, BBS7, and BBS9 manifest as more critical for the function and development of kidneys than peripheral subunits BBS1, BBS4, and BBS8/TTC8, suggesting that incomplete BBSome retains residual function at least in the kidney.

• MeSH
• ADP-ribosylační faktory genetika   MeSH
• Bardetův-Biedlův syndrom diagnóza   genetika   MeSH
• fenotyp * MeSH
• genetická predispozice k nemoci * MeSH
• genetické asociační studie * metody   MeSH
• genotyp * MeSH
• kognitivní dysfunkce genetika   MeSH
• ledviny abnormality   MeSH
• lidé MeSH
• mutace MeSH
• nemoci ledvin vrozené   genetika   MeSH
• penetrance MeSH
• proteiny genetika   MeSH
• vrozené vady genetika   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• metaanalýza MeSH
• práce podpořená grantem MeSH

Many plant viruses are vectored by insects in a persistent circulative manner. The insect gut and salivary gland are important barriers limiting virus spread, but the mechanisms by which viruses are able to cross the gut escape barriers of the insect remain largely unknown. Wheat dwarf virus (WDV), transmitted by Psammotettix alienus in a persistent, circulative, and nonpropagative manner, causes the most economically important virus disease in wheat. In this study, ADP ribosylation factor 1 (ARF1) was found to interact with the coat protein of WDV in a yeast two-hybrid, pull-down assay and to colocalise with virions in the gut and salivary glands of P. alienus. When transcription of ARF1 was suppressed by RNA interference, the WDV titre decreased in the haemolymph and salivary glands, and transmission efficiency decreased, but titre in the gut did not differ from that of the control. These data suggest that ARF1 of P. alienus binds to the WDV virion and helps virus spread from gut to haemolymph. Our study provides direct experimental evidence that WDV can use the existing membrane trafficking mechanism to aid its spread within the insect vector. This first analysis of the molecular interaction between WDV and its vector P. alienus contributes to understanding the mechanisms involved in circulative transmission of the virus by the leafhopper vector.

• MeSH
• ADP-ribosylační faktor 1 genetika   metabolismus   MeSH
• buněčné linie MeSH
• Geminiviridae patogenita   MeSH
• Hemiptera genetika   metabolismus   virologie   MeSH
• hmyz - vektory genetika   MeSH
• nemoci rostlin virologie   MeSH
• RNA interference MeSH
• slinné žlázy metabolismus   virologie   MeSH
• střeva virologie   MeSH
• techniky dvojhybridového systému MeSH
• virion metabolismus   MeSH
• virové plášťové proteiny metabolismus   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Membrane traffic at the trans-Golgi network (TGN) is crucial for correctly distributing various membrane proteins to their destination. Polarly localized auxin efflux proteins, including PIN-FORMED1 (PIN1), are dynamically transported between the endosomes and the plasma membrane (PM) in the plant cells. The intracellular trafficking of PIN1 protein is sensitive to the fungal toxin brefeldin A (BFA), which is known to inhibit guanine nucleotide exchange factors for ADP ribosylation factors (ARF GEFs) such as GNOM. However, the molecular details of the BFA-sensitive trafficking pathway have not been fully revealed. In a previous study, we identified an Arabidopsis mutant BFA-visualized endocytic trafficking defective 3 (ben3) which exhibited reduced sensitivity to BFA in terms of BFA-induced intracellular PIN1 agglomeration. Here, we show that BEN3 encodes a member of BIG family ARF GEFs, BIG2. BEN3/BIG2 tagged with fluorescent proteins co-localized with markers for the TGN/early endosome (EE). Inspection of conditionally induced de novo synthesized PIN1 confirmed that its secretion to the PM is BFA sensitive, and established BEN3/BIG2 as a crucial component of this BFA action at the level of the TGN/EE. Furthermore, ben3 mutation alleviated BFA-induced agglomeration of another TGN-localized ARF GEF, BEN1/MIN7. Taken together, our results suggest that BEN3/BIG2 is an ARF GEF component, which confers BFA sensitivity to the TGN/EE in Arabidopsis.

• MeSH
• ADP-ribosylační faktory genetika   metabolismus   MeSH
• alely MeSH
• Arabidopsis účinky léků   metabolismus   MeSH
• brefeldin A farmakologie   MeSH
• buněčná membrána účinky léků   metabolismus   MeSH
• endozomy účinky léků   metabolismus   MeSH
• fenotyp MeSH
• klonování DNA MeSH
• kompartmentace buňky MeSH
• nesmyslný kodon genetika   MeSH
• proteiny huseníčku genetika   metabolismus   MeSH
• semenáček účinky léků   růst a vývoj   MeSH
• trans-Golgiho síť účinky léků   metabolismus   MeSH
• transport proteinů účinky léků   MeSH
• výměnné faktory guaninnukleotidů metabolismus   MeSH
• zelené fluorescenční proteiny metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH

Exosomes are small vesicles that are secreted by cells and act as mediators of cell to cell communication. Because of their potential therapeutic significance, important efforts are being made towards characterizing exosomal contents. However, little is known about the mechanisms that govern exosome biogenesis. We have recently shown that the exosomal protein syntenin supports exosome production. Here we identify the small GTPase ADP ribosylation factor 6 (ARF6) and its effector phospholipase D2 (PLD2) as regulators of syntenin exosomes. ARF6 and PLD2 affect exosomes by controlling the budding of intraluminal vesicles (ILVs) into multivesicular bodies (MVBs). ARF6 also controls epidermal growth factor receptor degradation, suggesting a role in degradative MVBs. Yet ARF6 does not affect HIV-1 budding, excluding general effects on Endosomal Sorting Complexes Required for Transport. Our study highlights a novel pathway controlling ILV budding and exosome biogenesis and identifies an unexpected role for ARF6 in late endosomal trafficking.

• MeSH
• ADP-ribosylační faktory genetika   metabolismus   MeSH
• buněčné linie MeSH
• endozomální třídící komplexy pro transport genetika   metabolismus   MeSH
• erbB receptory metabolismus   MeSH
• exozómy enzymologie   genetika   metabolismus   MeSH
• fosfolipasa D genetika   metabolismus   MeSH
• HIV infekce genetika   metabolismus   virologie   MeSH
• HIV-1 fyziologie   MeSH
• lidé MeSH
• multivezikulární tělíska enzymologie   genetika   metabolismus   MeSH
• proteiny buněčného cyklu genetika   metabolismus   MeSH
• proteiny vázající vápník genetika   metabolismus   MeSH
• synteniny genetika   metabolismus   MeSH
• transport proteinů MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH

The delivery of newly synthesized soluble lysosomal hydrolases to the endosomal system is essential for lysosome function and cell homeostasis. This process relies on the proper trafficking of the mannose 6-phosphate receptors (MPRs) between the trans-Golgi network (TGN), endosomes and the plasma membrane. Many transmembrane proteins regulating diverse biological processes ranging from virus production to the development of multicellular organisms also use these pathways. To explore how cell signaling modulates MPR trafficking, we used high-throughput RNA interference (RNAi) to target the human kinome and phosphatome. Using high-content image analysis, we identified 127 kinases and phosphatases belonging to different signaling networks that regulate MPR trafficking and/or the dynamic states of the subcellular compartments encountered by the MPRs. Our analysis maps the MPR trafficking pathways based on enzymes regulating phosphatidylinositol phosphate metabolism. Furthermore, it reveals how cell signaling controls the biogenesis of post-Golgi tubular carriers destined to enter the endosomal system through a SRC-dependent pathway regulating ARF1 and RAC1 signaling and myosin II activity.

• MeSH
• ADP-ribosylační faktor 1 genetika   metabolismus   MeSH
• buněčná membrána enzymologie   MeSH
• endozomy enzymologie   MeSH
• fosfatidylinositolfosfáty metabolismus   MeSH
• genové regulační sítě MeSH
• HeLa buňky MeSH
• lidé MeSH
• mapy interakcí proteinů MeSH
• rac1 protein vázající GTP genetika   metabolismus   MeSH
• receptor IGF typ 2 genetika   metabolismus   MeSH
• regulace genové exprese enzymů MeSH
• RNA interference * MeSH
• shluková analýza MeSH
• signální transdukce MeSH
• skupina kinas odvozených od src-genu genetika   metabolismus   MeSH
• trans-Golgiho síť enzymologie   MeSH
• transfekce MeSH
• transport proteinů genetika   MeSH
• vysoce účinné nukleotidové sekvenování metody   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• audiovizuální média MeSH
• časopisecké články MeSH
• práce podpořená grantem MeSH