title: Control of Amino Acid Homeostasis by a Ubiquitin Ligase-Coactivator Protein Complex.
abstract: Intercellular amino acid transport is essential for the growth of all multicellular organisms, and its dysregulation is implicated in developmental disorders. By an unknown mechanism, amino acid efflux is stimulated in plants by overexpression of a membrane-localized protein (GLUTAMINEDUMPER 1 (GDU1)) that requires a ubiquitin ligase (LOSS OF GDU 2(LOG2). Here we further explore the physiological consequences of the interaction between these two proteins. LOG2 ubiquitin ligase activity is necessary for GDU1-dependent tolerance to exogenous amino acids, and LOG2 self-ubiquitination was markedly stimulated by the GDU1 cytosolic domain, suggesting that GDU1 functions as an adaptor or coactivator of amino acid exporter(s). However, other consequences more typical of a ligase-substrate relationship are observed: disruption of theLOG2gene increased thein vivohalf-life of GDU1, mass spectrometry confirmed that LOG2 ubiquitinates GDU1 at cytosolic lysines, and GDU1 protein levels decreased upon co-expression with active, but not enzymatically inactive LOG2. Altogether these data indicate LOG2 negatively regulates GDU1 protein accumulation by a mechanism dependent upon cytosolic GDU1 lysines. Although GDU1-lysine substituted protein exhibited diminishedin vivoubiquitination, overexpression of GDU1 lysine mutants still conferred amino acid tolerance in a LOG2-dependent manner, consistent with GDU1 being both a substrate and facilitator of LOG2 function. From these data, we offer a model in which GDU1 activates LOG2 to stimulate amino acid export, a process that could be negatively regulated by GDU1 ubiquitination and LOG2 self-ubiquitination.
authors.affiliation: From the Department of Molecular and Cellular Biology, University of California, Davis, Davis, California 95616 and.
authors.forename: Damian
authors.initials: D
authors.lastname: Guerra
authors.affiliation: From the Department of Molecular and Cellular Biology, University of California, Davis, Davis, California 95616 and.
authors.forename: Sonia M
authors.initials: SM
authors.lastname: Chapiro
authors.affiliation: Department of Plant Pathology, Physiology, and Weed Science, Virginia Polytechnic Institute and State University, Blacksburg, Virginia 24061.
authors.forename: Réjane
authors.initials: R
authors.lastname: Pratelli
authors.affiliation: Department of Plant Pathology, Physiology, and Weed Science, Virginia Polytechnic Institute and State University, Blacksburg, Virginia 24061.
authors.forename: Shi
authors.initials: S
authors.lastname: Yu
authors.affiliation: From the Department of Molecular and Cellular Biology, University of California, Davis, Davis, California 95616 and.
authors.forename: Weitao
authors.initials: W
authors.lastname: Jia
authors.affiliation: From the Department of Molecular and Cellular Biology, University of California, Davis, Davis, California 95616 and.
authors.forename: Julie
authors.initials: J
authors.lastname: Leary
authors.affiliation: Department of Plant Pathology, Physiology, and Weed Science, Virginia Polytechnic Institute and State University, Blacksburg, Virginia 24061.
authors.forename: Guillaume
authors.identifier: 0000-0001-7520-1059
authors.initials: G
authors.lastname: Pilot
authors.affiliation: From the Department of Molecular and Cellular Biology, University of California, Davis, Davis, California 95616 and jcallis@ucdavis.edu.
authors.forename: Judy
authors.identifier: 0000-0002-0622-078X
authors.initials: J
authors.lastname: Callis
mesh_terms: D000596:Amino Acids / Q000737:chemistry*; D017360:Arabidopsis / Q000235:genetics / Q000378:metabolism; D029681:Arabidopsis Proteins / Q000378:metabolism*; D002853:Chromatography, Liquid; D003433:Crosses, Genetic; D003600:Cytosol / Q000378:metabolism; D025461:Feedback, Physiological; D006706:Homeostasis; D008239:Lysine / Q000737:chemistry; D008565:Membrane Proteins / Q000378:metabolism*; D010641:Phenotype; D000072417:Protein Domains; D053719:Tandem Mass Spectrometry; D014026:Nicotiana / Q000235:genetics; D044767:Ubiquitin-Protein Ligases / Q000378:metabolism*; D054875:Ubiquitination
chemical_list: D000596:Amino Acids; D029681:Arabidopsis Proteins; C492757:GDU1 protein, Arabidopsis; D008565:Membrane Proteins; C586318:Log2 protein, Arabidopsis; D044767:Ubiquitin-Protein Ligases; D008239:Lysine
keywords: E3 ubiquitin ligase; Loss of GDU1 2; amino acid resistance; amino acid transport; glutamine dumper; protein degradation; ubiquitin; ubiquitin ligase; ubiquitylation (ubiquitination)