When yeast cells growing on a poor nitrogen source are supplied with NH4+ ions, several nitrogen permeases including the general amino acid permease (Gap1p) are rapidly and completely inactivated. This report shows that inactivation by NH4+ of the Gap1 permease is accompanied by its degradation. A functional NPl1 gene product is required for both inactivation and degradation of Gap1p. Molecular analysis of the NPl1 gene showed that it is identical to RSP5. The RSP5 product is a ubiquitin-protein ligase (E3 enzyme) whose physiological function was, however, unknown. Its C-terminal region is very similar to that of other members of the E6-AP-like family of ubiquitin-protein ligases. Its N-terminal region contains a single C2 domain that may be a Ca(2+)-dependent phospholipid interaction motif, followed by several copies of a recently identified domain called WW(P). The Npi1/Rsp5 protein has a homologue both in humans and in mice, the latter being involved in brain development. Stress-induced degradation of the uracil permease (Fur4p), a process in which ubiquitin is probably involved, was also found to require a functional NPl1/RSP5 product. Chromosomal deletion of NPl1/RSP5 showed that this gene is essential for cell viability. In the viable npi1/rsp5 strain, expression of NPl1/RSP5 is reduced as a result of insertion of a Ty1 element in its 5' region. Our results show that the Npi1/Rsp5 ubiquitin-protein ligase participates in induced degradation of at least two permeases, Gap1p and Fur4p, and probably also other proteins.
Transport of 4-aminobutyric acid (GABA) in Saccharomyces cerevisiae is mediated by three transport systems: the general amino acid permease (GAP1 gene), the proline permease (PUT4 gene), and a specific GABA permease (UGA4 gene) which is induced in the presence of GABA. The UGA4 gene encoding the inducible GABA-specific transporter was cloned and sequenced and its expression analyzed. The predicted amino acid sequence shows that UGA4 encodes a 62 kDa protein having 9-12 putative membrane-spanning regions. The predicted UGA4 protein shares significant sequence similarity with the yeast choline transporter (CTR gene), exhibiting but limited similarity to the previously reported GABA transporters, i.e. the yeast GAP1 and PUT4 permeases and the rat brain GAT-1 transporter. Induction of UGA4 in the presence of GABA is exerted at the level of UGA4 mRNA accumulation, most probably at the level of transcription itself. This induction is conferred by the 5' flanking region and requires the integrity of two positive regulatory proteins, the inducer-specific factor UGA3 and the pleiotropic factor UGA35/DURL/DAL81. In the absence of the pleiotropic UGA43/DAL80 repressor, UGA4 is constitutively expressed at high level.
SummaryThe general amino acid permease, Gap1, of Saccharomyces cerevisiae is very active in cells grown on proline as the sole nitrogen source. Adding NH 4 þ 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 þ -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 ␣-factor receptor, Ste2p. Another replacement, substitution of two alanines for a di-leucine motif, likewise protects the Gap1 permease against NH 4 þ -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 phosphor ylation 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 þ 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 þ -insensitive permeases, namely the arginine (Can1p) and uracil (Fur4p) permeases.
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