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

Mol Microbiol. 1997 May;24(3):607-16. doi: 10.1046/j.1365-2958.1997.3771735.x.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Amino Acid Transport Systems
  • Base Sequence
  • Cloning, Molecular
  • DNA Primers / genetics
  • DNA, Fungal / genetics
  • Enzyme Inhibitors / pharmacology
  • Leucine / chemistry
  • Membrane Transport Modulators*
  • Membrane Transport Proteins / antagonists & inhibitors*
  • Membrane Transport Proteins / genetics*
  • Membrane Transport Proteins / metabolism
  • Molecular Sequence Data
  • Point Mutation
  • Quaternary Ammonium Compounds / pharmacology*
  • Saccharomyces cerevisiae / enzymology*
  • Sequence Deletion
  • Sequence Homology, Amino Acid

Substances

  • Amino Acid Transport Systems
  • DNA Primers
  • DNA, Fungal
  • Enzyme Inhibitors
  • Membrane Transport Modulators
  • Membrane Transport Proteins
  • Quaternary Ammonium Compounds
  • Leucine