title: The ubiquitin E3 ligase LOSS OF GDU2 is required for GLUTAMINE DUMPER1-induced amino acid secretion in Arabidopsis.
abstract: Amino acids serve as transport forms for organic nitrogen in the plant, and multiple transport steps are involved in cellular import and export. While the nature of the export mechanism is unknown, overexpression of GLUTAMINE DUMPER1 (GDU1) in Arabidopsis (Arabidopsis thaliana) led to increased amino acid export. To gain insight into GDU1's role, we searched for ethyl-methanesulfonate suppressor mutants and performed yeast-two-hybrid screens. Both methods uncovered the same gene, LOSS OF GDU2 (LOG2), which encodes a RING-type E3 ubiquitin ligase. The interaction between LOG2 and GDU1 was confirmed by glutathione S-transferase pull-down, in vitro ubiquitination, and in planta coimmunoprecipitation experiments. Confocal microscopy and subcellular fractionation indicated that LOG2 and GDU1 both localized to membranes and were enriched at the plasma membrane. LOG2 expression overlapped with GDU1 in the xylem and phloem tissues of Arabidopsis. The GDU1 protein encoded by the previously characterized intragenic suppressor mutant log1-1, with an arginine in place of a conserved glycine, failed to interact in the multiple assays, suggesting that the Gdu1D phenotype requires the interaction of GDU1 with LOG2. This hypothesis was supported by suppression of the Gdu1D phenotype after reduction of LOG2 expression using either artificial microRNAs or a LOG2 T-DNA insertion. Altogether, in accordance with the emerging bulk of data showing membrane protein regulation via ubiquitination, these data suggest that the interaction of GDU1 and the ubiquitin ligase LOG2 plays a significant role in the regulation of amino acid export from plant cells.
authors.affiliation: Department of Plant Pathology, Physiology, and Weed Science, Virginia Tech, Blacksburg, Virginia 24061, USA.
authors.forename: Réjane
authors.initials: R
authors.lastname: Pratelli
authors.forename: Damian D
authors.initials: DD
authors.lastname: Guerra
authors.forename: Shi
authors.initials: S
authors.lastname: Yu
authors.forename: Mark
authors.initials: M
authors.lastname: Wogulis
authors.forename: Edward
authors.initials: E
authors.lastname: Kraft
authors.forename: Wolf B
authors.initials: WB
authors.lastname: Frommer
authors.forename: Judy
authors.initials: J
authors.lastname: Callis
authors.forename: Guillaume
authors.initials: G
authors.lastname: Pilot
mesh_terms: D019943:Amino Acid Substitution / Q000235:genetics; D000596:Amino Acids / Q000378:metabolism*; D017360:Arabidopsis / Q000201:enzymology*; D029681:Arabidopsis Proteins / Q000737:chemistry / Q000378:metabolism*; D002462:Cell Membrane / Q000201:enzymology; D004269:DNA, Bacterial / Q000235:genetics; D016153:Genes, Suppressor; D005966:Glucuronidase / Q000378:metabolism; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D035683:MicroRNAs / Q000378:metabolism; D008861:Microsomes / Q000201:enzymology; D009154:Mutation / Q000235:genetics; D019814:Myristic Acid / Q000378:metabolism; D010641:Phenotype; D058526:Plant Vascular Bundle / Q000201:enzymology; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D013347:Subcellular Fractions / Q000201:enzymology; D013489:Suppression, Genetic; D044767:Ubiquitin-Protein Ligases / Q000737:chemistry / Q000378:metabolism*; D054875:Ubiquitination
chemical_list: D000596:Amino Acids; D029681:Arabidopsis Proteins; D004269:DNA, Bacterial; C492757:GDU1 protein, Arabidopsis; D008565:Membrane Proteins; D035683:MicroRNAs; C036483:T-DNA; D019814:Myristic Acid; C586318:Log2 protein, Arabidopsis; D044767:Ubiquitin-Protein Ligases; D005966:Glucuronidase