vesicle coats
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Clathrin-mediated endocytosis (CME) is key to maintaining the transmembrane protein composition of cells' limiting membranes. During mammalian CME, a reversible phosphorylation event occurs on Thr156 of the μ2 subunit of the main endocytic clathrin adaptor, AP2. We show that this phosphorylation event starts during clathrin-coated pit (CCP) initiation and increases throughout CCP lifetime. μ2Thr156 phosphorylation favors a new, cargo-bound conformation of AP2 and simultaneously creates a binding platform for the endocytic NECAP proteins but without significantly altering AP2's cargo affinity in vitro. We describe the structural bases of both. NECAP arrival at CCPs parallels that of clathrin and increases with μ2Thr156 phosphorylation. In turn, NECAP recruits drivers of late stages of CCP formation, including SNX9, via a site distinct from where NECAP binds AP2. Disruption of the different modules of this phosphorylation-based temporal regulatory system results in CCP maturation being delayed and/or stalled, hence impairing global rates of CME.
- MeSH
- adaptorový proteinový komplex - alfa-podjednotky genetika MeSH
- adaptorový proteinový komplex 2 genetika metabolismus MeSH
- endocytóza genetika MeSH
- fosforylace genetika MeSH
- klathrin genetika metabolismus MeSH
- klathrinové vezikuly genetika metabolismus MeSH
- lidé MeSH
- potažené jamky v buněčné membráně genetika metabolismus MeSH
- třídící nexiny genetika MeSH
- vazba proteinů genetika MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
The eukaryotic phylum Parabasalia is composed primarily of anaerobic, endobiotic organisms such as the veterinary parasite Tritrichomonas foetus and the human parasite Trichomonas vaginalis, the latter causing the most prevalent, non-viral, sexually transmitted disease world-wide. Although a parasitic lifestyle is generally associated with a reduction in cell biology, T. vaginalis provides a striking counter-example. The 2007 T. vaginalis genome paper reported a massive and selective expansion of encoded proteins involved in vesicle trafficking, particularly those implicated in the late secretory and endocytic systems. Chief amongst these were the hetero-tetrameric adaptor proteins or 'adaptins', with T. vaginalis encoding ∼3.5 times more such proteins than do humans. The provenance of such a complement, and how it relates to the transition from a free-living or endobiotic state to parasitism, remains unclear. In this study, we performed a comprehensive bioinformatic and molecular evolutionary investigation of the heterotetrameric cargo adaptor-derived coats, comparing the molecular complement and evolution of these proteins between T. vaginalis, T. foetus and the available diversity of endobiotic parabasalids. Notably, with the recent discovery of Anaeramoeba spp. as the free-living sister lineage to all parabasalids, we were able to delve back to time points earlier in the lineage's history than ever before. We found that, although T. vaginalis still encodes the most HTAC subunits amongst parabasalids, the duplications giving rise to the complement took place more deeply and at various stages across the lineage. While some duplications appear to have convergently shaped the parasitic lineages, the largest jump is in the transition from free-living to endobiotic lifestyle with both gains and losses shaping the encoded complement. This work details the evolution of a cellular system across an important lineage of parasites and provides insight into the evolutionary dynamics of an example of expansion of protein machinery, counter to the more common trends observed in many parasitic systems.
- MeSH
- lidé MeSH
- Parabasalidea * MeSH
- paraziti * MeSH
- Trichomonas vaginalis * genetika MeSH
- Tritrichomonas foetus * genetika MeSH
- výpočetní biologie MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
The Arf GTPase controls formation of the COPI vesicle coat. Recent structural models of COPI revealed the positioning of two Arf1 molecules in contrasting molecular environments. Each of these pockets for Arf1 is expected to also accommodate an Arf GTPase-activating protein (ArfGAP). Structural evidence and protein interactions observed between isolated domains indirectly suggest that each niche preferentially recruits one of the two ArfGAPs known to affect COPI, i.e. Gcs1/ArfGAP1 and Glo3/ArfGAP2/3, although only partial structures are available. The functional role of the unique non-catalytic domain of either ArfGAP has not been integrated into the current COPI structural model. Here, we delineate key differences in the consequences of triggering GTP hydrolysis through the activity of one versus the other ArfGAP. We demonstrate that Glo3/ArfGAP2/3 specifically triggers Arf1 GTP hydrolysis impinging on the stability of the COPI coat. We show that the Snf1 kinase complex, the yeast homologue of AMP-activated protein kinase (AMPK), phosphorylates the region of Glo3 that is crucial for this effect and, thereby, regulates its function in the COPI-vesicle cycle. Our results revise the model of ArfGAP function in the molecular context of COPI.This article has an associated First Person interview with the first author of the paper.
- MeSH
- biologické modely * MeSH
- COP-vezikuly genetika metabolismus MeSH
- obalový proteinový komplex I genetika metabolismus MeSH
- proteiny aktivující GTPasu genetika metabolismus MeSH
- Saccharomyces cerevisiae - proteiny genetika metabolismus MeSH
- Saccharomyces cerevisiae genetika metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Močové extracelulární vezikuly (uEVs) představují slibný nástroj pro neinvazivní diagnostiku a monitorování onemocnění ledvin. Tyto vezikuly, secernované buňkami ledvin, obsahují biomolekuly odrážející stav mateřských buněk. Výzkum se zaměřuje na využití uEVs jako biomarkerů pro chronická onemocnění ledvin, akutní poškození ledvin, diabetickou nefropatii a monitorování po transplantaci ledvin. Kromě diagnostického potenciálu jsou uEVs zkoumány pro terapeutické aplikace v regeneraci ledvinové tkáně. Přes výzvy v standardizaci izolačních a analytických metod uEVs, pokrok ve vývoji metod charakterizace uEVs podporuje jejich klinické využití.
Urinary extracellular vesicles (uEVs) represent a promising tool for non-invasive diagnosis and monitoring of kidney disease. These vesicles, secreted by kidney cells, contain biomolecules reflecting the status of the parent cells. Research focuses on using uEVs as biomarkers for chronic kidney disease, acute kidney injury, diabetic nephropathy and monitoring kidney transplantation. In addition to diagnostic potential, uEVs are being investigated for therapeutic applications in kidney tissue regeneration. Despite challenges in standardizing uEVs isolation and analysis methods, progress in the development of uEVs characterization methods supports their clinical use.
- Klíčová slova
- močové extracelulární vezikuly,
- MeSH
- biologické markery * analýza MeSH
- COP-vezikuly fyziologie MeSH
- lidé MeSH
- nemoci ledvin * diagnóza patofyziologie MeSH
- urogenitální systém anatomie a histologie MeSH
- Check Tag
- lidé MeSH
Giardia intestinalis is a globally important microbial pathogen with considerable public health, agricultural, and economic burden. Genome sequencing and comparative analyses have elucidated G. intestinalis to be a taxonomically diverse species consisting of at least eight different sub-types (assemblages A-H) that can infect a great variety of animal hosts, including humans. The best studied of these are assemblages A and B which have a broad host range and have zoonotic transmissibility towards humans where clinical Giardiasis can range from asymptomatic to diarrheal disease. Epidemiological surveys as well as previous molecular investigations have pointed towards critical genomic level differences within numerous molecular pathways and families of parasite virulence factors within assemblage A and B isolates. In this study, we explored the necessary machinery for the formation of vesicles and cargo transport in 89 Canadian isolates of assemblage A and B G. intestinalis. Considerable variability within the molecular complement of the endolysosomal ESCRT protein machinery, adaptor coat protein complexes, and ARF regulatory system have previously been reported. Here, we confirm inter-assemblage, but find no intra-assemblage variation within the trafficking systems examined. This variation includes losses of subunits belonging to the ESCRTIII as well as novel lineage specific duplications in components of the COPII machinery, ARF1, and ARFGEF families (BIG and CYTH). Since differences in disease manifestation between assemblages A and B have been controversially reported, our findings may well have clinical implications and even taxonomic, as the membrane trafficking system underpin parasite survival, pathogenesis, and propagation.
- MeSH
- feces parazitologie MeSH
- genomika MeSH
- genotyp MeSH
- Giardia lamblia * MeSH
- giardiáza * parazitologie MeSH
- lidé MeSH
- veřejné zdravotnictví MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- Geografické názvy
- Kanada MeSH
Monovalent-cation homeostasis, crucial for all living cells, is ensured by the activity of various types of ion transport systems located either in the plasma membrane or in the membranes of organelles. A key prerequisite for the functioning of ion-transporting proteins is their proper trafficking to the target membrane. The cornichon family of COPII cargo receptors is highly conserved in eukaryotic cells. By simultaneously binding their cargoes and a COPII-coat subunit, cornichons promote the incorporation of cargo proteins into the COPII vesicles and, consequently, the efficient trafficking of cargoes via the secretory pathway. In this review, we summarize current knowledge about cornichon proteins (CNIH/Erv14), with an emphasis on yeast and mammalian cornichons and their role in monovalent-cation homeostasis. Saccharomyces cerevisiae cornichon Erv14 serves as a cargo receptor of a large portion of plasma-membrane proteins, including several monovalent-cation transporters. By promoting the proper targeting of at least three housekeeping ion transport systems, Na+, K+/H+ antiporter Nha1, K+ importer Trk1 and K+ channel Tok1, Erv14 appears to play a complex role in the maintenance of alkali-metal-cation homeostasis. Despite their connection to serious human diseases, the repertoire of identified cargoes of mammalian cornichons is much more limited. The majority of current information is about the structure and functioning of CNIH2 and CNIH3 as auxiliary subunits of AMPAR multi-protein complexes. Based on their unique properties and easy genetic manipulation, we propose yeast cells to be a useful tool for uncovering a broader spectrum of human cornichons ́ cargoes.
- MeSH
- COP-vezikuly metabolismus MeSH
- homeostáza fyziologie MeSH
- iontový transport fyziologie MeSH
- lidé MeSH
- membránové proteiny metabolismus MeSH
- proteiny přenášející kationty metabolismus MeSH
- Saccharomyces cerevisiae - proteiny metabolismus genetika MeSH
- Saccharomyces cerevisiae * metabolismus MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- přehledy MeSH
Erv14, a conserved cargo receptor of COPII vesicles, helps the proper trafficking of many but not all transporters to the yeast plasma membrane, for example, three out of five alkali-metal-cation transporters in Saccharomyces cerevisiae. Among them, the Nha1 cation/proton antiporter, which participates in cell cation and pH homeostasis, is a large membrane protein (985 aa) possessing a long hydrophilic C-terminus (552 aa) containing six conserved regions (C1-C6) with unknown function. A short Nha1 version, lacking almost the entire C-terminus, still binds to Erv14 but does not need it to be targeted to the plasma membrane. Comparing the localization and function of ScNha1 variants shortened at its C-terminus in cells with or without Erv14 reveals that only ScNha1 versions possessing the complete C5 region are dependent on Erv14. In addition, our broad evolutionary conservation analysis of fungal Na+ /H+ antiporters identified new conserved regions in their C-termini, and our experiments newly show C5 and other, so far unknown, regions of the C-terminus, to be involved in the functionality and substrate specificity of ScNha1. Taken together, our results reveal that also relatively small hydrophilic parts of some yeast membrane proteins underlie their need to interact with the Erv14 cargo receptor.
- MeSH
- antiportéry genetika metabolismus MeSH
- buněčná membrána metabolismus MeSH
- COP-vezikuly genetika metabolismus MeSH
- endoplazmatické retikulum metabolismus MeSH
- membránové proteiny metabolismus fyziologie MeSH
- proteiny přenášející kationty metabolismus MeSH
- Saccharomyces cerevisiae - proteiny genetika metabolismus fyziologie MeSH
- Saccharomyces cerevisiae metabolismus MeSH
- sodík metabolismus MeSH
- transport proteinů MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Vesicles bearing either cationic (amino) groups or zwitterionic (amino acid) groups on the surface were coated with a reactive multivalent hydrophilic N-(2-hydroxypropyl)methacrylamide polymer (PHPMA) and its positively charged analogue (3 mol % quaternary ammonium groups), both having reactive thiazolidine-2-thione (TT) groups randomly distributed along the polymer chain. The vesicles were dispersed in water at a concentration of 1 mg/mL. The effect of surface charges of model vesicles on the surface coating efficiency was evaluated. The changes in the weight-average molecular weight, in the hydrodynamic size, and in the zeta-potential of model vesicles were tested using light scattering methods. The most effective coating of vesicles was observed for the zwitterionic vesicles coated with the positively charged hydrophilic PHPMA-TT copolymer at a concentration of reactive polymer cp = 2 mg/mL. The coating efficiency was more than 1 order of magnitude higher than that obtained for positively charged vesicles coated by the uncharged hydrophilic polymer at the same cp.
The export of membrane proteins along the secretory pathway is initiated at the endoplasmic reticulum after proteins are folded and packaged inside this organelle by their recruiting into the coat complex COPII vesicles. It is proposed that cargo receptors are required for the correct transport of proteins to its target membrane, however, little is known about ER export signals for cargo receptors. Erv14/Cornichon belong to a well conserved protein family in Eukaryotes, and have been proposed to function as cargo receptors for many transmembrane proteins. Amino acid sequence alignment showed the presence of a conserved acidic motif in the C-terminal in homologues from plants and yeast. Here, we demonstrate that mutation of the C-terminal acidic motif from ScErv14 or OsCNIH1, did not alter the localization of these cargo receptors, however it modified the proper targeting of the plasma membrane transporters Nha1p, Pdr12p and Qdr2p. Our results suggest that mistargeting of these plasma membrane proteins is a consequence of a weaker interaction between the cargo receptor and cargo proteins caused by the mutation of the C-terminal acidic motif.
- MeSH
- ABC transportéry genetika MeSH
- aminokyselinové motivy genetika MeSH
- buněčná membrána genetika metabolismus MeSH
- COP-vezikuly genetika metabolismus MeSH
- endoplazmatické retikulum genetika metabolismus MeSH
- Golgiho aparát genetika metabolismus MeSH
- membránové proteiny genetika metabolismus MeSH
- membránové transportní proteiny genetika MeSH
- Na(+)-H(+) antiport genetika MeSH
- rýže (rod) genetika MeSH
- Saccharomyces cerevisiae - proteiny genetika MeSH
- Saccharomyces cerevisiae genetika MeSH
- sbalování proteinů MeSH
- sekvence aminokyselin genetika MeSH
- sekvenční seřazení MeSH
- transport proteinů genetika MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
