title: Proteasomal modulation of cellular SNAT2 (SLC38A2) abundance and function by unsaturated fatty acid availability.
abstract: Expression and activity of the System A/SNAT2 (SLC38A2) amino acid transporter is up-regulated by amino acid starvation and hypertonicity by a mechanism dependent on both ATF4-mediated transcription of the SLC38A2 gene and enhanced stabilization of SNAT2 itself, which forms part of an integrated cellular stress response to nutrient deprivation and osmotic stress. Here we demonstrate that this adaptive increase in System A function is restrained in cells subjected to prior incubation with linoleic acid (LOA, an unsaturated C18:2 fatty acid) for 24 h. While fatty acid treatment had no detectable effect upon stress-induced SNAT2 or ATF4 gene transcription, the associated increase in SNAT2 protein/membrane transport activity were strongly suppressed in L6 myotubes or HeLa cells preincubated with LOA. Cellular ubiquitination of many proteins was increased by LOA and although the fatty acid-induced loss of SNAT2 could be attenuated by proteasomal inhibition, the functional increase in System A transport activity associated with amino acid starvation/hypertonicity that depends upon processing/maturation and delivery of SNAT2 to the cell surface could not be rescued. LOA up-regulated cellular expression of Nedd4.2, an E3-ligase implicated in SNAT2 ubiquitination, but shRNA-directed Nedd4.2 gene silencing could not curb fatty acid-induced loss of SNAT2 adaptation. However, expression of SNAT2 in which seven putative lysyl-ubiquitination sites in the cytoplasmic N-terminal domain were mutated to alanine protected SNAT2 against LOA-induced proteasomal degradation. Collectively, our findings indicate that increased availability of unsaturated fatty acids can compromise the stress-induced induction/adaptation in SNAT2 expression and function by promoting its degradation via the ubiquitin-proteasome system.
authors: From the Division of Cell Signalling and Immunology, Sir James Black Centre, College of Life Sciences, University of Dundee, Dundee, DD1 5EH, United Kingdom.
authors: Francesca
authors: F
authors: Nardi
authors: From the Division of Cell Signalling and Immunology, Sir James Black Centre, College of Life Sciences, University of Dundee, Dundee, DD1 5EH, United Kingdom.
authors: Thorsten M
authors: TM
authors: Hoffmann
authors: From the Division of Cell Signalling and Immunology, Sir James Black Centre, College of Life Sciences, University of Dundee, Dundee, DD1 5EH, United Kingdom.
authors: Clare
authors: C
authors: Stretton
authors: From the Division of Cell Signalling and Immunology, Sir James Black Centre, College of Life Sciences, University of Dundee, Dundee, DD1 5EH, United Kingdom.
authors: Emma
authors: E
authors: Cwiklinski
authors: From the Division of Cell Signalling and Immunology, Sir James Black Centre, College of Life Sciences, University of Dundee, Dundee, DD1 5EH, United Kingdom.
authors: Peter M
authors: PM
authors: Taylor
authors: From the Division of Cell Signalling and Immunology, Sir James Black Centre, College of Life Sciences, University of Dundee, Dundee, DD1 5EH, United Kingdom h.s.hundal@dundee.ac.uk.
authors: Harinder S
authors: HS
authors: Hundal
mesh_terms: D026924:Amino Acid Transport System A / Q000378:metabolism*; D000818:Animals; D056827:Endosomal Sorting Complexes Required for Transport / Q000235:genetics / Q000378:metabolism; D057809:HEK293 Cells; D006367:HeLa Cells; D006801:Humans; D019787:Linoleic Acid / Q000502:physiology*; D018485:Muscle Fibers, Skeletal / Q000378:metabolism; D000075702:Nedd4 Ubiquitin Protein Ligases; D009997:Osmotic Pressure; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism*; D051381:Rats; D014158:Transcription, Genetic; D044767:Ubiquitin-Protein Ligases / Q000235:genetics / Q000378:metabolism; D015854:Up-Regulation
chemical_list: D026924:Amino Acid Transport System A; D056827:Endosomal Sorting Complexes Required for Transport; C498797:SLC38A2 protein, human; D019787:Linoleic Acid; C000618479:NEDD4L protein, rat; D000075702:Nedd4 Ubiquitin Protein Ligases; C000618483:Nedd4 protein, human; C000618484:Nedd4 protein, rat; C000618485:Nedd4L protein, human; D044767:Ubiquitin-Protein Ligases; D046988:Proteasome Endopeptidase Complex
keywords: ATF4; Amino acid transporter; Linoleic acid; SNAT2; amino acid transport; fatty acid; hypertonicity; muscle; proteasome; ubiquitin
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