Eukaryotic cells possess considerable internal complexity, differentiating them from prokaryotes. Eukaryogenesis, an evolutionary transitional period culminating in the last eukaryotic common ancestor (LECA), marked the origin of the eukaryotic endomembrane system. LECA is reconstructed as possessing intracellular complexity akin to modern eukaryotes. Construction of endomembrane compartments involved three key gene families: coatomer, BAR-domain proteins, and ESCRT. Each has a distinct evolutionary origin, but of these coatomer and BAR proteins are eukaryote specific, while ESCRT has more ancient origins. We discuss the structural motifs defining these three membrane-coating complexes and suggest that compared with BAR and ESCRT, the coatomer architecture had a unique ability to be readily and considerably modified, unlocking functional diversity and enabling the development of the eukaryotic cell.

• MeSH
• biologická evoluce MeSH
• COP-vezikuly MeSH
• endozomální třídící komplexy pro transport metabolismus   MeSH
• Eukaryota * genetika   MeSH
• eukaryotické buňky * metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH
• Názvy látek
• endozomální třídící komplexy pro transport MeSH

Vacuolar proteins play essential roles in plant physiology and development, but the factors and the machinery regulating their vesicle trafficking through the endomembrane compartments remain largely unknown. We and others have recently identified an evolutionarily conserved plant endosomal sorting complex required for transport (ESCRT)-associated protein apoptosis-linked gene-2 interacting protein X (ALIX), which plays canonical functions in the biogenesis of the multivesicular body/prevacuolar compartment (MVB/PVC) and in the sorting of ubiquitinated membrane proteins. In this study, we elucidate the roles and underlying mechanism of ALIX in regulating vacuolar transport of soluble proteins, beyond its conventional ESCRT function in eukaryotic cells. We show that ALIX colocalizes and physically interacts with the retromer core subunits Vps26 and Vps29 in planta. Moreover, double-mutant analysis reveals the genetic interaction of ALIX with Vps26 and Vps29 for regulating trafficking of soluble vacuolar proteins. Interestingly, depletion of ALIX perturbs membrane recruitment of Vps26 and Vps29 and alters the endosomal localization of vacuolar sorting receptors (VSRs). Taken together, ALIX functions as a unique retromer core subcomplex regulator by orchestrating receptor-mediated vacuolar sorting of soluble proteins.

• Klíčová slova
• ESCRT machiner, endosomal recycling, multivesicular body/prevacuolar compartment (MVB/PVC), retromer complex, vacuolar trafficking,
• MeSH
• Arabidopsis * metabolismus   MeSH
• endozomální třídící komplexy pro transport metabolismus   MeSH
• endozomy metabolismus   MeSH
• proteiny huseníčku * genetika   metabolismus   MeSH
• rostliny metabolismus   MeSH
• transport proteinů fyziologie   MeSH
• transportní proteiny metabolismus   MeSH
• vakuoly metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• ALIX protein, Arabidopsis MeSH Prohlížeč
• endozomální třídící komplexy pro transport MeSH
• proteiny huseníčku * MeSH
• transportní proteiny MeSH

Neural precursor cells expressed developmentally downregulated protein 4-2 (Nedd4-2), a homologous to the E6-AP carboxyl terminus (HECT) ubiquitin ligase, triggers the endocytosis and degradation of its downstream target molecules by regulating signal transduction through interactions with other targets, including 14-3-3 proteins. In our previous study, we found that 14-3-3 binding induces a structural rearrangement of Nedd4-2 by inhibiting interactions between its structured domains. Here, we used time-resolved fluorescence intensity and anisotropy decay measurements, together with fluorescence quenching and mass spectrometry, to further characterize interactions between Nedd4-2 and 14-3-3 proteins. The results showed that 14-3-3 binding affects the emission properties of AEDANS-labeled WW3, WW4, and, to a lesser extent, WW2 domains, and reduces their mobility, but not those of the WW1 domain, which remains mobile. In contrast, 14-3-3 binding has the opposite effect on the active site of the HECT domain, which is more solvent exposed and mobile in the complexed form than in the apo form of Nedd4-2. Overall, our results suggest that steric hindrance of the WW3 and WW4 domains combined with conformational changes in the catalytic domain may account for the 14-3-3 binding-mediated regulation of Nedd4-2.

• MeSH
• endozomální třídící komplexy pro transport * metabolismus   MeSH
• katalytická doména MeSH
• nervové kmenové buňky * metabolismus   MeSH
• proteiny 14-3-3 metabolismus   MeSH
• ubikvitinligasy Nedd4 metabolismus   MeSH
• ubikvitinligasy metabolismus   MeSH
• vazba proteinů MeSH
• WW domény MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• endozomální třídící komplexy pro transport * MeSH
• proteiny 14-3-3 MeSH
• ubikvitinligasy Nedd4 MeSH
• ubikvitinligasy MeSH

Cytokinetic membrane abscission is a spatially and temporally regulated process that requires ESCRT (endosomal sorting complexes required for transport)–dependent control of membrane remodeling at the midbody, a subcellular organelle that defines the cleavage site. Alteration of ESCRT function can lead to cataract, but the underlying mechanism and its relation to cytokinesis are unclear. We found a lens-specific cytokinetic process that required PI3K-C2α (phosphatidylinositol-4-phosphate 3-kinase catalytic subunit type 2α), its lipid product PI(3,4)P2 (phosphatidylinositol 3,4-bisphosphate), and the PI(3,4)P2–binding ESCRT-II subunit VPS36 (vacuolar protein-sorting-associated protein 36). Loss of each of these components led to impaired cytokinesis, triggering premature senescence in the lens of fish, mice, and humans. Thus, an evolutionarily conserved pathway underlies the cell type–specific control of cytokinesis that helps to prevent early onset cataract by protecting from senescence.

• MeSH
• biologická evoluce MeSH
• buněčné linie MeSH
• cytokineze * MeSH
• dánio pruhované MeSH
• endozomální třídící komplexy pro transport metabolismus   MeSH
• fosfatidylinositol-3-kinasy genetika   metabolismus   MeSH
• fosfatidylinositol-4,5-difosfát metabolismus   MeSH
• fosfatidylinositoly metabolismus   MeSH
• katarakta metabolismus   patologie   MeSH
• lidé MeSH
• mutace MeSH
• myši MeSH
• oční čočka cytologie   růst a vývoj   metabolismus   MeSH
• předčasné stárnutí MeSH
• proteiny buněčného cyklu metabolismus   MeSH
• proteiny dánia pruhovaného genetika   metabolismus   MeSH
• proteiny vázající vápník metabolismus   MeSH
• stárnutí buněk * MeSH
• tubulin metabolismus   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH
• Názvy látek
• CHMP4B protein, human MeSH Prohlížeč
• endozomální třídící komplexy pro transport MeSH
• fosfatidylinositol-4,5-difosfát MeSH
• fosfatidylinositoly MeSH
• PDCD6IP protein, human MeSH Prohlížeč
• phosphoinositide-3,4-bisphosphate MeSH Prohlížeč
• PIK3C2A protein, human MeSH Prohlížeč
• Pik3c2a protein, mouse MeSH Prohlížeč
• proteiny buněčného cyklu MeSH
• proteiny dánia pruhovaného MeSH
• proteiny vázající vápník MeSH
• tubulin MeSH
• VPS36 protein, human MeSH Prohlížeč

BACKGROUND: Comparing a parasitic lineage to its free-living relatives is a powerful way to understand how that evolutionary transition to parasitism occurred. Giardia intestinalis (Fornicata) is a leading cause of gastrointestinal disease world-wide and is famous for its unusual complement of cellular compartments, such as having peripheral vacuoles instead of typical endosomal compartments. Endocytosis plays an important role in Giardia's pathogenesis. Endosomal sorting complexes required for transport (ESCRT) are membrane-deforming proteins associated with the late endosome/multivesicular body (MVB). MVBs are ill-defined in G. intestinalis, and roles for identified ESCRT-related proteins are not fully understood in the context of its unique endocytic system. Furthermore, components thought to be required for full ESCRT functionality have not yet been documented in this species. RESULTS: We used genomic and transcriptomic data from several Fornicata species to clarify the evolutionary genome streamlining observed in Giardia, as well as to detect any divergent orthologs of the Fornicata ESCRT subunits. We observed differences in the ESCRT machinery complement between Giardia strains. Microscopy-based investigations of key components of ESCRT machinery such as GiVPS36 and GiVPS25 link them to peripheral vacuoles, highlighting these organelles as simplified MVB equivalents. Unexpectedly, we show ESCRT components associated with the endoplasmic reticulum and, for the first time, mitosomes. Finally, we identified the rare ESCRT component CHMP7 in several fornicate representatives, including Giardia and show that contrary to current understanding, CHMP7 evolved from a gene fusion of VPS25 and SNF7 domains, prior to the last eukaryotic common ancestor, over 1.5 billion years ago. CONCLUSIONS: Our findings show that ESCRT machinery in G. intestinalis is far more varied and complete than previously thought, associates to multiple cellular locations, and presents changes in ESCRT complement which pre-date adoption of a parasitic lifestyle.

• Klíčová slova
• ESCRT, Endomembrane, Evolutionary Cell Biology, Excavata, Giardia, PV, Parasitism,
• MeSH
• biologická evoluce MeSH
• endozomální třídící komplexy pro transport * genetika   metabolismus   MeSH
• endozomy metabolismus   MeSH
• Giardia lamblia * genetika   metabolismus   MeSH
• transport proteinů MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• endozomální třídící komplexy pro transport * MeSH

The integration of complementary molecular methods (including X-ray crystallography, NMR spectroscopy, small angle X-ray/neutron scattering, and computational techniques) is frequently required to obtain a comprehensive understanding of dynamic macromolecular complexes. In particular, these techniques are critical for studying intrinsically disordered protein regions (IDRs) or intrinsically disordered proteins (IDPs) that are part of large protein:protein complexes. Here, we explain how to prepare IDP samples suitable for study using NMR spectroscopy, and describe a novel SAXS modeling method (ensemble refinement of SAXS; EROS) that integrates the results from complementary methods, including crystal structures and NMR chemical shift perturbations, among others, to accurately model SAXS data and describe ensemble structures of dynamic macromolecular complexes.

• Klíčová slova
• EROS, Ensemble, Intrinsically disordered proteins (IDP), NMR spectroscopy, SAXS,
• MeSH
• endozomální třídící komplexy pro transport chemie   metabolismus   MeSH
• konformace proteinů MeSH
• krystalografie rentgenová metody   MeSH
• lidé MeSH
• magnetická rezonanční spektroskopie metody   MeSH
• mitogenem aktivované proteinkinasy chemie   metabolismus   MeSH
• molekulární modely MeSH
• radiační rozptyl * MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH
• Názvy látek
• endozomální třídící komplexy pro transport MeSH
• mitogenem aktivované proteinkinasy MeSH

Receptor tyrosine kinases (RTKs) form multiprotein complexes that initiate and propagate intracellular signals and determine the RTK-specific signalling patterns. Unravelling the full complexity of protein interactions within the RTK-associated complexes is essential for understanding of RTK functions, yet it remains an understudied area of cell biology. We describe a comprehensive approach to characterize RTK interactome. A single tag immunoprecipitation and phosphotyrosine protein isolation followed by mass-spectrometry was used to identify proteins interacting with fibroblast growth factor receptor 3 (FGFR3). A total of 32 experiments were carried out in two different cell types and identified 66 proteins out of which only 20 (30.3%) proteins were already known FGFR interactors. Using co-immunoprecipitations, we validated FGFR3 interaction with adapter protein STAM1, transcriptional regulator SHOX2, translation elongation factor eEF1A1, serine/threonine kinases ICK, MAK and CCRK, and inositol phosphatase SHIP2. We show that unappreciated signalling mediators exist for well-studied RTKs, such as FGFR3, and may be identified via proteomic approaches described here. These approaches are easily adaptable to other RTKs, enabling identification of novel signalling mediators for majority of the known human RTKs.

• Klíčová slova
• FGFR3, Fibroblast growth factor, interactome, receptor tyrosine kinase, signal transduction,
• MeSH
• adaptorové proteiny signální transdukční genetika   metabolismus   MeSH
• buňky NIH 3T3 MeSH
• cyklin-dependentní kinasy genetika   metabolismus   MeSH
• elongační faktor 1 genetika   metabolismus   MeSH
• endozomální třídící komplexy pro transport genetika   metabolismus   MeSH
• fosfatidylinositol-3,4,5-trisfosfát-5-fosfatasy genetika   metabolismus   MeSH
• fosfoproteiny genetika   metabolismus   MeSH
• fosforylace MeSH
• HEK293 buňky MeSH
• homeodoménové proteiny genetika   metabolismus   MeSH
• kinasa aktivující cyklin dependentní kinasy MeSH
• lidé MeSH
• mapování interakce mezi proteiny MeSH
• myši MeSH
• protein-serin-threoninkinasy genetika   metabolismus   MeSH
• proteomika metody   MeSH
• receptor fibroblastových růstových faktorů, typ 3 genetika   metabolismus   MeSH
• regulace genové exprese * MeSH
• signální transdukce genetika   MeSH
• stanovení celkové genové exprese MeSH
• vazba proteinů MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• adaptorové proteiny signální transdukční MeSH
• CILK1 protein, human MeSH Prohlížeč
• cyklin-dependentní kinasy MeSH
• EEF1A1 protein, human MeSH Prohlížeč
• elongační faktor 1 MeSH
• endozomální třídící komplexy pro transport MeSH
• FGFR3 protein, human MeSH Prohlížeč
• fosfatidylinositol-3,4,5-trisfosfát-5-fosfatasy MeSH
• fosfoproteiny MeSH
• homeodoménové proteiny MeSH
• INPPL1 protein, human MeSH Prohlížeč
• kinasa aktivující cyklin dependentní kinasy MeSH
• MAK protein, human MeSH Prohlížeč
• protein-serin-threoninkinasy MeSH
• receptor fibroblastových růstových faktorů, typ 3 MeSH
• SHOX2 protein, human MeSH Prohlížeč
• STAM protein, human MeSH Prohlížeč

Endosomal sorting complexes required for transport (ESCRTs) are involved in the formation of multivesicular bodies and sorting of targeted proteins to the yeast vacuole. The deletion of seven genes encoding components of the ESCRT machinery render Saccharomyces cerevisiae cells sensitive to high extracellular CaCl2 concentrations as well as to low pH in media. In this work, we focused on intracellular pH (pHin) homeostasis of these mutants. None of the studied ESCRT mutants exhibited an altered pHin level compared to the wild type under standard growth conditions. Nevertheless, 60 min of CaCl2 treatment resulted in a more significant drop in pHin levels in these mutants than in the wild type, suggesting that pHin homeostasis is affected in ESCRT mutants upon the addition of calcium. Similarly, CaCl2 treatment caused a bigger pHin decrease in cells lacking the vacuolar Ca(2+)/H(+) antiporter Vcx1 which indicates a role for this protein in the maintenance of proper pHin homeostasis when cells need to cope with a high CaCl2 concentration in media. Importantly, ESCRT gene deletions in the vcx1Δ strain did not result in an increase in the CaCl2-invoked drop in the pHin levels of cells, which demonstrates a genetic interaction between VCX1 and studied ESCRT genes.

• Klíčová slova
• ESCRT, Vcx1, calcium, pHin,
• MeSH
• antiportéry metabolismus   MeSH
• delece genu MeSH
• endozomální třídící komplexy pro transport genetika   metabolismus   MeSH
• fyziologický stres * MeSH
• homeostáza * MeSH
• koncentrace vodíkových iontů MeSH
• Saccharomyces cerevisiae - proteiny metabolismus   MeSH
• Saccharomyces cerevisiae účinky léků   genetika   růst a vývoj   fyziologie   MeSH
• vápník metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• antiportéry MeSH
• endozomální třídící komplexy pro transport MeSH
• Saccharomyces cerevisiae - proteiny MeSH
• vápník MeSH
• VCX1 protein, S cerevisiae MeSH Prohlížeč

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 faktor 6 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
• Názvy látek
• ADP-ribosylační faktor 6 MeSH
• ADP-ribosylační faktory MeSH
• ARF6 protein, human MeSH Prohlížeč
• endozomální třídící komplexy pro transport MeSH
• erbB receptory MeSH
• fosfolipasa D MeSH
• PDCD6IP protein, human MeSH Prohlížeč
• phospholipase D2 MeSH Prohlížeč
• proteiny buněčného cyklu MeSH
• proteiny vázající vápník MeSH
• synteniny MeSH