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.
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.
Hein
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C
Laboratoire de Physiologie Cellulaire et de Génétique des Levures, Université Libre de Bruxelles, Belgium.
André
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B
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
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