Fluorescence imaging-based screen identifies ARF GEF component of early endosomal trafficking.
Endocytic vesicle trafficking is crucial for regulating activity and localization of plasma membrane components, but the process is still poorly genetically defined in plants. Membrane proteins of the PIN-FORMED (PIN) family exhibit polar localization in plant cells and facilitate cellular efflux of the plant hormone auxin, thereby regulating multiple developmental processes. PIN proteins undergo constitutive endocytosis and GNOM ARF GEF-dependent recycling, and their localization is under extensive regulation by developmental and environmental cues. We designed a fluorescence imaging-based screen to identify Arabidopsis thaliana mutants defective in internalization of proteins including PINs from the plasma membrane. We identified three mutant loci, BFA-visualized endocytic trafficking defective1 (ben1) through ben3 that do not efficiently accumulate PIN1-GFP in intracellular compartments after inhibition of recycling and secretion by fungal toxin brefeldin A (BFA). Fine mapping revealed that BEN1 encodes an ARF GEF vesicle trafficking regulator from the functionally uncharacterized BIG class. ben1 mutant has been previously implicated in pathogen response and shows cell polarity, BFA sensitivity, and growth defects. BEN1 is involved in endocytosis of plasma membrane proteins and localizes to early endocytic compartments distinct from GNOM-positive endosomes. Our results identify BEN1 as the ARF GEF mediating early endosomal traffic.
Tanaka
Hirokazu
H
Department of Plant Systems Biology, Flanders Institute for Biotechnology (VIB) and Department of Molecular Genetics, Ghent University, 9052 Gent, Belgium.
Kitakura
Saeko
S
De Rycke
Riet
R
De Groodt
Ruth
R
Friml
Jirí
J
D020727:ADP-Ribosylation Factors / Q000235:genetics / Q000378:metabolism*; D000429:Alcohol Oxidoreductases / Q000235:genetics / Q000378:metabolism*; D017360:Arabidopsis / Q000166:cytology / Q000235:genetics / Q000502:physiology; D029681:Arabidopsis Proteins / Q000235:genetics / Q000378:metabolism*; D001692:Biological Transport / Q000502:physiology; D016764:Cell Polarity; D011992:Endosomes / Q000378:metabolism*; D020662:Guanine Nucleotide Exchange Factors / Q000235:genetics / Q000378:metabolism*; D026901:Membrane Transport Proteins / Q000235:genetics / Q000378:metabolism; D009154:Mutation; D018517:Plant Roots / Q000166:cytology / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D021601:trans-Golgi Network / Q000378:metabolism / Q000648:ultrastructure
D029681:Arabidopsis Proteins; D020662:Guanine Nucleotide Exchange Factors; D026901:Membrane Transport Proteins; C116363:PIN1 protein, Arabidopsis; D011993:Recombinant Fusion Proteins; D000429:Alcohol Oxidoreductases; C523325:BEN1 protein, Arabidopsis; D020727:ADP-Ribosylation Factors