A C‐terminal di‐leucine motif and nearby sequences are required for NH 4 + ‐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 NH 4 + 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 NH 4 + ‐induced inactivation. An in vivo isolated mutation ( gap1 pgr  ) causes a single Glu→Lys substitution in an amino acid context similar to the DXKSS sequence involved in ubiquitination and endocytosis of the yeast α‐factor receptor, Ste2p. Another replacement, substitution of two alanines for a di‐leucine motif, likewise protects the Gap1 permease against NH 4 + ‐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 NH 4 + 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 NH 4 + ‐insensitive permeases, namely the arginine (Can1p) and uracil (Fur4p) permeases.