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
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
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
