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

The fibroblast growth factor receptors (FGFRs) are important oncogenes promoting tumor progression in many types of cancer, such as breast, bladder, and lung cancer as well as multiple myeloma and rhabdomyosarcoma. However, little is known about how these receptors are internalized and down-regulated in cells. We have here applied proximity biotin labeling to identify proteins involved in FGFR4 signaling and trafficking. For this purpose we fused a mutated biotin ligase, BirA*, to the C-terminal tail of FGFR4 (FGFR4-BirA*) and the fusion protein was stably expressed in U2OS cells. Upon addition of biotin to these cells, proteins in proximity to the FGFR4-BirA* fusion protein became biotinylated and could be isolated and identified by quantitative mass spectrometry. We identified in total 291 proteins, including 80 proteins that were enriched in samples where the receptor was activated by the ligand (FGF1), among them several proteins previously found to be involved in FGFR signaling (e.g., FRS2, PLCγ, RSK2 and NCK2). Interestingly, many of the identified proteins were implicated in endosomal transport, and by precise annotation we were able to trace the intracellular pathways of activated FGFR4. Validating the data by confocal and three-dimensional structured illumination microscopy analysis, we concluded that FGFR4 uses clathrin-mediated endocytosis for internalization and is further sorted from early endosomes to the recycling compartment and the trans-Golgi network. Depletion of cells for clathrin heavy chain led to accumulation of FGFR4 at the cell surface and increased levels of active FGFR4 and PLCγ, while AKT and ERK signaling was diminished, demonstrating that functional clathrin-mediated endocytosis is required for proper FGFR4 signaling. Thus, this study reveals proteins and pathways involved in FGFR4 transport and signaling that provide possible targets and opportunities for therapeutic intervention in FGFR4 aberrant cancer.

• MeSH
• barvení a značení MeSH
• biotinylace MeSH
• endocytóza MeSH
• endozomy metabolismus   MeSH
• klathrin metabolismus   MeSH
• lidé MeSH
• mikroskopie metody   MeSH
• nádorové buněčné linie MeSH
• receptor fibroblastových růstových faktorů, typ 4 metabolismus   MeSH
• signální transdukce MeSH
• trans-Golgiho síť metabolismus   MeSH
• transport proteinů MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

PI4K IIalpha is a critical enzyme for the maintenance of Golgi and is also known to function in the synaptic vesicles. The product of its catalytical function, phosphatidylinositol 4-phosphate (PI4P), is an important lipid molecule because it is a hallmark of the Golgi and TGN, is directly recognized by many proteins and also serves as a precursor molecule for synthesis of higher phosphoinositides. Here, we report crystal structures of PI4K IIalpha enzyme in the apo-state and inhibited by calcium. The apo-structure reveals a surprising rigidity of the active site residues important for catalytic activity. The structure of calcium inhibited kinase reveals how calcium locks ATP in the active site.

• MeSH
• adenosintrifosfát metabolismus   MeSH
• fosfatidylinositolfosfáty metabolismus   MeSH
• fosfotransferasy s alkoholovou skupinou jako akceptorem antagonisté a inhibitory   chemie   MeSH
• katalytická doména účinky léků   MeSH
• konformace proteinů MeSH
• krystalografie rentgenová MeSH
• molekulární modely MeSH
• trans-Golgiho síť účinky léků   metabolismus   MeSH
• vápník farmakologie   MeSH
• vazba proteinů MeSH
• vztahy mezi strukturou a aktivitou MeSH
• Publikační typ
• časopisecké články MeSH

In land plants polar auxin transport is one of the substantial processes guiding whole plant polarity and morphogenesis. Directional auxin fluxes are mediated by PIN auxin efflux carriers, polarly localized at the plasma membrane. The polarization of exocytosis in yeast and animals is assisted by the exocyst: an octameric vesicle-tethering complex and an effector of Rab and Rho GTPases. Here we show that rootward polar auxin transport is compromised in roots of Arabidopsis thaliana loss-of-function mutants in the EXO70A1 exocyst subunit. The recycling of PIN1 and PIN2 proteins from brefeldin-A compartments is delayed after the brefeldin-A washout in exo70A1 and sec8 exocyst mutants. Relocalization of PIN1 and PIN2 proteins after prolonged brefeldin-A treatment is largely impaired in these mutants. At the same time, however, plasma membrane localization of GFP:EXO70A1, and the other exocyst subunits studied (GFP:SEC8 and YFP:SEC10), is resistant to brefeldin-A treatment. In root cells of the exo70A1 mutant, a portion of PIN2 is internalized and retained in specific, abnormally enlarged, endomembrane compartments that are distinct from VHA-a1-labelled early endosomes or the trans-Golgi network, but are RAB-A5d positive. We conclude that the exocyst is involved in PIN1 and PIN2 recycling, and thus in polar auxin transport regulation.

• MeSH
• Arabidopsis cytologie   účinky léků   genetika   metabolismus   MeSH
• biologický transport MeSH
• brefeldin A farmakologie   MeSH
• buněčná membrána metabolismus   MeSH
• endozomy metabolismus   MeSH
• epidermis rostlin cytologie   účinky léků   genetika   metabolismus   MeSH
• fenotyp MeSH
• geneticky modifikované rostliny MeSH
• kořeny rostlin cytologie   účinky léků   genetika   metabolismus   MeSH
• kyseliny indoloctové metabolismus   MeSH
• membránové transportní proteiny genetika   metabolismus   MeSH
• mutace MeSH
• proteiny huseníčku genetika   metabolismus   MeSH
• rekombinantní fúzní proteiny MeSH
• rozvržení tělního plánu MeSH
• semenáček cytologie   účinky léků   genetika   metabolismus   MeSH
• trans-Golgiho síť metabolismus   MeSH
• vezikulární transportní proteiny genetika   metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, U.S. Gov't, Non-P.H.S. MeSH

LY294002 is a synthetic quercetin-like compound, which, unlike wortmannin, is more specific inhibitor of phosphatidylinositol 3-kinase (PI3K). It inhibits endocytosis and vacuolar transport. We report here on the proteome-wide effects of LY294002 on Arabidopsis roots focusing on proteins involved in vesicular trafficking and stress response. At the subcellular level, LY294002 caused swelling and clustering of late endosomes leading to inhibition of vacuolar transport. At the proteome level, this compound caused changes in abundances of proteins categorized to 10 functional classes. Among proteins involved in vesicular trafficking, a small GTPase ARFA1f was more abundant, indicating its possible contribution to the aggregation and fusion of late endosomes triggered by LY294002. Our study provides new information on storage proteins and vacuolar hydrolases in vegetative tissues treated by LY294002. Vacuolar hydrolases were downregulated, while storage proteins were more abundant, suggesting that storage proteins were protected from degradation in swollen multivesicular bodies upon LY294002 treatment. Upregulation of 2S albumin was validated by immunoblotting and immunolabeling analyses. Our study also pointed to the control of antioxidant enzyme machinery by PI3K because LY294002 downregulated two isozymes of superoxide dismutase. This most likely occurred via PI3K-mediated downregulation of protein AtDJ1A. Finally, we discuss specificity differences of LY294002 and wortmannin against PI3K, which are reflected at the proteome level. Compared with wortmannin, LY294002 showed more narrow and perhaps also more specific effects on proteins, as suggested by gene ontology functional annotation.

• MeSH
• anotace sekvence MeSH
• Arabidopsis cytologie   účinky léků   metabolismus   MeSH
• biologický transport MeSH
• chromony farmakologie   MeSH
• fosfatidylinositol-3-kinasy metabolismus   MeSH
• fyziologický stres MeSH
• inhibitory fosfoinositid-3-kinasy MeSH
• kořeny rostlin cytologie   účinky léků   metabolismus   MeSH
• morfoliny farmakologie   MeSH
• proteiny huseníčku antagonisté a inhibitory   genetika   metabolismus   MeSH
• proteom genetika   metabolismus   MeSH
• regulace genové exprese u rostlin účinky léků   MeSH
• trans-Golgiho síť metabolismus   MeSH
• transportní vezikuly metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• Research Support, N.I.H., Extramural MeSH

Wortmannin is a widely used pharmaceutical compound which is employed to define vesicular trafficking routes of particular proteins or cellular compounds. It targets phosphatidylinositol 3-kinase and phosphatidylinositol 4-kinases in a dose-dependent manner leading to the inhibition of protein vacuolar sorting and endocytosis. Combined proteomics and cell biological approaches have been used in this study to explore the effects of wortmannin on Arabidopsis root cells, especially on proteome and endomembrane trafficking. On the subcellular level, wortmannin caused clustering, fusion, and swelling of trans-Golgi network (TGN) vesicles and multivesicular bodies (MVBs) leading to the formation of wortmannin-induced multivesicular compartments. Appearance of wortmannin-induced compartments was associated with depletion of TGN as revealed by electron microscopy. On the proteome level, wortmannin induced massive changes in protein abundance profiles. Wortmannin-sensitive proteins belonged to various functional classes. An inhibition of vacuolar trafficking by wortmannin was related to the downregulation of proteins targeted to the vacuole, as showed for vacuolar proteases. A small GTPase, RabA1d, which regulates vesicular trafficking at TGN, was identified as a new protein negatively affected by wortmannin. In addition, Sec14 was upregulated and PLD1 alpha was downregulated by wortmannin.

• MeSH
• androstadieny farmakologie   MeSH
• Arabidopsis cytologie   účinky léků   metabolismus   MeSH
• fosfolipasa D metabolismus   MeSH
• kořeny rostlin cytologie   účinky léků   metabolismus   MeSH
• multivezikulární tělíska účinky léků   metabolismus   MeSH
• proteiny huseníčku metabolismus   MeSH
• proteiny přenášející fosfolipidy metabolismus   MeSH
• proteom metabolismus   MeSH
• rab proteiny vázající GTP metabolismus   MeSH
• trans-Golgiho síť účinky léků   metabolismus   ultrastruktura   MeSH
• transport proteinů účinky léků   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH