title: Internal amino acids promote Gap1 permease ubiquitylation via TORC1/Npr1/14-3-3-dependent control of the Bul arrestin-like adaptors.
abstract: Ubiquitylation of many plasma membrane proteins promotes their endocytosis followed by degradation in the lysosome. The yeast general amino acid permease, Gap1, is ubiquitylated and downregulated when a good nitrogen source like ammonium is provided to cells growing on a poor nitrogen source. This ubiquitylation requires the Rsp5 ubiquitin ligase and the redundant arrestin-like Bul1 and Bul2 adaptors. Previous studies have shown that Gap1 ubiquitylation involves the TORC1 kinase complex, which inhibits the Sit4 phosphatase. This causes inactivation of the protein kinase Npr1, which protects Gap1 against ubiquitylation. However, the mechanisms inducing Gap1 ubiquitylation after Npr1 inactivation remain unknown. We here show that on a poor nitrogen source, the Bul adaptors are phosphorylated in an Npr1-dependent manner and bound to 14-3-3 proteins that protect Gap1 against downregulation. After ammonium is added and converted to amino acids, the Bul proteins are dephosphorylated, dissociate from the 14-3-3 proteins, and undergo ubiquitylation. Furthermore, dephosphorylation of Bul requires the Sit4 phosphatase, which is essential to Gap1 downregulation. The data support the emerging concept that permease ubiquitylation results from activation of the arrestin-like adaptors of the Rsp5 ubiquitin ligase, this coinciding with their dephosphorylation, dissociation from the inhibitory 14-3-3 proteins, and ubiquitylation.
authors.affiliation: Molecular Physiology of the Cell, Université Libre de Bruxelles, IBMM, Gosselies, Belgium.
authors.forename: Ahmad
authors.initials: A
authors.lastname: Merhi
authors.forename: Bruno
authors.initials: B
authors.lastname: André
mesh_terms: D048948:14-3-3 Proteins / Q000235:genetics / Q000378:metabolism*; D048868:Adaptor Proteins, Signal Transducing / Q000235:genetics / Q000378:metabolism*; D026905:Amino Acid Transport Systems / Q000235:genetics / Q000378:metabolism*; D000596:Amino Acids / Q000378:metabolism; D015966:Gene Expression Regulation, Fungal; D008247:Lysosomes / Q000378:metabolism; D010766:Phosphorylation; D011494:Protein Kinases / Q000235:genetics / Q000378:metabolism*; D059748:Proteolysis; D000644:Quaternary Ammonium Compounds / Q000378:metabolism / Q000494:pharmacology; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction / Q000235:genetics; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*; D025801:Ubiquitin / Q000378:metabolism; D044767:Ubiquitin-Protein Ligases / Q000235:genetics / Q000378:metabolism*; D054875:Ubiquitination
chemical_list: D048948:14-3-3 Proteins; D048868:Adaptor Proteins, Signal Transducing; D026905:Amino Acid Transport Systems; D000596:Amino Acids; C099939:BUL1 protein, S cerevisiae; C117205:BUL2 protein, S cerevisiae; C491145:GAP1 protein, S cerevisiae; D000644:Quaternary Ammonium Compounds; D029701:Saccharomyces cerevisiae Proteins; C561842:TORC1 protein complex, S cerevisiae; D014157:Transcription Factors; D025801:Ubiquitin; C066958:NPR1 protein, S cerevisiae; D044767:Ubiquitin-Protein Ligases; D011494:Protein Kinases