title: A C-terminal di-leucine motif and nearby sequences are required for NH4(+)-induced inactivation and degradation of the general amino acid permease, Gap1p, of Saccharomyces cerevisiae.
abstract: The general amino acid permease, Gap1, of Saccharomyces cerevisiae is very active in cells grown on proline as the sole nitrogen source. Adding NH4+ to the medium triggers inactivation and degradation of the permease via a regulatory process involving Npi1p/Rsp5p, a ubiquitin-protein ligase. In this study, we describe several mutations affecting the C-terminal region of Gap1p that render the permease resistant to NH4(+)-induced inactivation. An in vivo isolated mutation (gap1pgr) causes a single Glu-->Lys substitution in an amino acid context similar to the DXKSS sequence involved in ubiquitination and endocytosis of the yeast alpha-factor receptor, Ste2p. Another replacement, substitution of two alanines for a di-leucine motif, likewise protects the Gap1 permease against NH4(+)-induced inactivation. In mammalian cells, such a motif is involved in the internalization of several cell-surface proteins. These data provide the first indication that a di-leucine motif influences the function of a plasma membrane protein in yeast. Mutagenesis of a putative phosphorylation site upstream from the di-leucine motif altered neither the activity nor the regulation of the permease. In contrast, deletion of the last eleven amino acids of Gap1p, a region conserved in other amino acid permeases, conferred resistance to NH4+ inactivation. Although the C-terminal region of Gap1p plays an important role in nitrogen control of activity, it was not sufficient to confer this regulation to two NH4(+)-insensitive permeases, namely the arginine (Can1p) and uracil (Fur4p) permeases.
authors.affiliation: Laboratoire de Physiologie Cellulaire et de Génétique des Levures, Université Libre de Bruxelles, Belgium.
authors.forename: C
authors.initials: C
authors.lastname: Hein
authors.forename: B
authors.initials: B
authors.lastname: André
mesh_terms: D000595:Amino Acid Sequence; D026905:Amino Acid Transport Systems; D001483:Base Sequence; D003001:Cloning, Molecular; D017931:DNA Primers / Q000235:genetics; D004271:DNA, Fungal / Q000235:genetics; D004791:Enzyme Inhibitors / Q000494:pharmacology; D007930:Leucine / Q000737:chemistry; D049990:Membrane Transport Modulators; D026901:Membrane Transport Proteins / Q000037:antagonists & inhibitors* / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D017354:Point Mutation; D000644:Quaternary Ammonium Compounds / Q000494:pharmacology*; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D017384:Sequence Deletion; D017386:Sequence Homology, Amino Acid
chemical_list: D026905:Amino Acid Transport Systems; D017931:DNA Primers; D004271:DNA, Fungal; D004791:Enzyme Inhibitors; D049990:Membrane Transport Modulators; D026901:Membrane Transport Proteins; D000644:Quaternary Ammonium Compounds; D007930:Leucine