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.

• Klíčová slova
• ARF GEF, Arabidopsis, Auxin, Brefeldin A, PIN-FORMED1, trans-Golgi network,
• 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
• Názvy látek
• ADP-ribosylační faktory MeSH
• big2 protein Arabidopsis MeSH Prohlížeč
• brefeldin A MeSH
• nesmyslný kodon MeSH
• proteiny huseníčku MeSH
• výměnné faktory guaninnukleotidů MeSH
• zelené fluorescenční proteiny MeSH

BACKGROUND: Processes of anterograde and retrograde membrane trafficking play an important role in cellular homeostasis and dynamic rearrangements of the plasma membrane (PM) in all eukaryotes. These processes depend on the activity of adenosine ribosylation factors (ARFs), a family of GTP-binding proteins and their guanine exchange factors (GEFs). However, knowledge on the function and specificity of individual ARF-GEFs for individual steps of membrane trafficking pathways is still limited in plants. RESULTS: In this work, treatments with various trafficking inhibitors showed that the endocytosis of FM 4-64 is largely dynamin-dependent and relies on proteins containing endocytic tyrosine-based internalization motif and intact cytoskeleton. Interestingly, brefeldin A (BFA), reported previously as an inhibitor of anterograde membrane trafficking in plants, appeared to be the most potent inhibitor of endocytosis in tobacco. In concert with this finding, we demonstrate that the point mutation in the Sec7 domain of the GNOM-LIKE protein1a (NtGNL1a) confers intracellular trafficking pathway-specific BFA resistance. The internalization of FM 4-64 and trafficking of PIN-FORMED1 (PIN1) auxin efflux carrier in BY-2 tobacco cells were studied to reveal the function of the ARF-GEF NtGNL1a in these. CONCLUSIONS: Altogether, our observations uncovered the role of NtGNL1a in endocytosis, including endocytosis of PM proteins (as PIN1 auxin efflux carrier). Moreover these data emphasize the need of careful evaluation of mode of action of non-native inhibitors in various species. In addition, they demonstrate the potential of tobacco BY-2 cells for selective mapping of ARF-GEF-regulated endomembrane trafficking pathways.

• MeSH
• endocytóza MeSH
• kvartérní amoniové sloučeniny metabolismus   MeSH
• pyridinové sloučeniny metabolismus   MeSH
• rostlinné buňky fyziologie   MeSH
• rostlinné proteiny genetika   metabolismus   MeSH
• tabák genetika   fyziologie   MeSH
• transport proteinů MeSH
• výměnné faktory guaninnukleotidů genetika   metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• FM 4-64 MeSH Prohlížeč
• kvartérní amoniové sloučeniny MeSH
• pyridinové sloučeniny MeSH
• rostlinné proteiny MeSH
• výměnné faktory guaninnukleotidů MeSH