title: Identification of a Novel Regulatory Mechanism of Nutrient Transport Controlled by TORC1-Npr1-Amu1/Par32.
abstract: Fine-tuning the plasma-membrane permeability to essential nutrients is fundamental to cell growth optimization. Nutritional signals including nitrogen availability are integrated by the TORC1 complex which notably regulates arrestin-mediated endocytosis of amino-acid transporters. Ammonium is a ubiquitous compound playing key physiological roles in many, if not all, organisms. In yeast, it is a preferred nitrogen source transported by three Mep proteins which are orthologues of the mammalian Rhesus factors. By combining genetic, kinetic, biochemical and cell microscopy analyses, the current study reveals a novel mechanism enabling TORC1 to regulate the inherent activity of ammonium transport proteins, independently of arrestin-mediated endocytosis, identifying the still functional orphan Amu1/Par32 as a selective regulator intermediate. We show that, under poor nitrogen supply, the TORC1 effector kinase' Npr1' promotes phosphorylation of Amu1/Par32 which appears mainly cytosolic while ammonium transport proteins are active. Upon preferred nitrogen supplementation, like glutamine or ammonium addition, TORC1 upregulation enables Npr1 inhibition and Amu1/Par32 dephosphorylation. In these conditions, as in Npr1-lacking cells, hypophosphorylated Amu1/Par32 accumulates at the cell surface and mediates the inhibition of specific ammonium transport proteins. We show that the integrity of a conserved repeated motif of Amu1/Par32 is required for the interaction with these transport proteins. This study underscores the diversity of strategies enabling TORC1-Npr1 to selectively monitor cell permeability to nutrients by discriminating between transporters to be degraded or transiently inactivated and kept stable at the plasma membrane. This study further identifies the function of Amu1/Par32 in acute control of ammonium transport in response to variations in nitrogen availability.
authors.affiliation: Biology of Membrane Transport, IBMM, Université Libre de Bruxelles, Gosselies, Belgium.
authors.forename: Mélanie
authors.initials: M
authors.lastname: Boeckstaens
authors.affiliation: Biology of Membrane Transport, IBMM, Université Libre de Bruxelles, Gosselies, Belgium.
authors.forename: Ahmad
authors.initials: A
authors.lastname: Merhi
authors.affiliation: Biology of Membrane Transport, IBMM, Université Libre de Bruxelles, Gosselies, Belgium.
authors.forename: Elisa
authors.initials: E
authors.lastname: Llinares
authors.affiliation: Biology of Membrane Transport, IBMM, Université Libre de Bruxelles, Gosselies, Belgium.
authors.forename: Pascale
authors.initials: P
authors.lastname: Van Vooren
authors.affiliation: IRIBHM, Université Libre de Bruxelles, Campus ERASME, Brussels, Belgium.
authors.forename: Jean-Yves
authors.initials: JY
authors.lastname: Springael
authors.affiliation: Laboratoire des Biopolymères et des nanomatériaux supramoléculaires, Université Libre de Bruxelles, Brussels, Belgium.
authors.forename: René
authors.initials: R
authors.lastname: Wintjens
authors.affiliation: Biology of Membrane Transport, IBMM, Université Libre de Bruxelles, Gosselies, Belgium.
authors.forename: Anna Maria
authors.initials: AM
authors.lastname: Marini
mesh_terms: D026905:Amino Acid Transport Systems / Q000235:genetics; D027682:Cation Transport Proteins / Q000378:metabolism; D002462:Cell Membrane / Q000502:physiology; D015966:Gene Expression Regulation, Fungal / Q000502:physiology; D046912:Multiprotein Complexes / Q000378:metabolism; D010539:Permeability; D010766:Phosphorylation / Q000235:genetics; D011494:Protein Kinases / Q000235:genetics / Q000378:metabolism*; D021381:Protein Transport / Q000235:genetics / Q000502:physiology*; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*
chemical_list: D026905:Amino Acid Transport Systems; D027682:Cation Transport Proteins; C088951:MEP1 protein, S cerevisiae; C107271:MEP3 protein, S cerevisiae; D046912:Multiprotein Complexes; C000607163:Par32 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; C561842:TORC1 protein complex, S cerevisiae; D014157:Transcription Factors; C066958:NPR1 protein, S cerevisiae; D011494:Protein Kinases