title: Regulation of amino acid transporter ATA2 by ubiquitin ligase Nedd4-2.
abstract: We report here that ubiquitin ligase Nedd4-2 regulates amino acid transporter ATA2 activity on the cell surface. We first found that a proteasome inhibitor MG132 increased the uptake of alpha-(methylamino)isobutyric acid, a model substrate for amino acid transport system A, in 3T3-L1 adipocytes as well as the preadipocytes. Transient expression of Nedd4-2 in Xenopus oocytes and Chinese hamster ovary cells down-regulated the ATA2 transport activity induced by injected cRNA and transfected cDNA, respectively. Neither the Nedd4-2 mutant with defective catalytic domain nor c-Cbl affected the ATA2 activity significantly. RNA-mediated interference of Nedd4-2 increased the ATA2 activity in the cells, and this was associated with decreased polyubiquitination of ATA2 on the cell surface membrane. Immunofluorescent analysis of Nedd4-2 in the adipocytes stably transfected with the enhanced green fluorescent protein (EGFP)-tagged ATA2 showed the co-localization of Nedd4-2 and EGFP-ATA2 in the plasma membrane but not in the perinuclear ATA2 storage site, supporting the idea that the primary site for the ubiquitination of ATA2 is the plasma membrane. These data suggest that ATA2 on the plasma membrane is subject to polyubiquitination by Nedd4-2 with consequent endocytotic sequestration and proteasomal degradation and that this process is an important determinant of the density of ATA2 functioning on the cell surface.
authors.affiliation: Mitsubishi Kagaku Institute of Life Sciences, 11 Minamiooya, Machida, Tokyo 194-8511, Japan.
authors.forename: Takahiro
authors.initials: T
authors.lastname: Hatanaka
authors.forename: Yasue
authors.initials: Y
authors.lastname: Hatanaka
authors.forename: Mitsutoshi
authors.initials: M
authors.lastname: Setou
mesh_terms: D041721:3T3-L1 Cells; D026924:Amino Acid Transport System A / Q000096:biosynthesis* / Q000737:chemistry; D000621:Aminoisobutyric Acids / Q000494:pharmacology; D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D056827:Endosomal Sorting Complexes Required for Transport; D005786:Gene Expression Regulation; D049452:Green Fluorescent Proteins / Q000494:pharmacology; D007976:Leupeptins / Q000494:pharmacology; D051379:Mice; D000075702:Nedd4 Ubiquitin Protein Ligases; D009865:Oocytes / Q000378:metabolism; D011480:Protease Inhibitors / Q000494:pharmacology; D025801:Ubiquitin / Q000737:chemistry / Q000378:metabolism; D044767:Ubiquitin-Protein Ligases / Q000096:biosynthesis / Q000737:chemistry / Q000502:physiology*; D014981:Xenopus; D029867:Xenopus Proteins
chemical_list: D026924:Amino Acid Transport System A; D000621:Aminoisobutyric Acids; D056827:Endosomal Sorting Complexes Required for Transport; D007976:Leupeptins; D011480:Protease Inhibitors; C498798:Slc38a2 protein, mouse; D025801:Ubiquitin; D029867:Xenopus Proteins; D049452:Green Fluorescent Proteins; C017911:2-(methylamino)isobutyric acid; D000075702:Nedd4 Ubiquitin Protein Ligases; C000618482:Nedd4 protein, Xenopus; C000618461:Nedd4l protein, mouse; C000618460:nedd4l protein, Xenopus; D044767:Ubiquitin-Protein Ligases; C072553:benzyloxycarbonylleucyl-leucyl-leucine aldehyde