Nitrogen-regulated Ubiquitination of the Gap1 Permease ofSaccharomyces cerevisiae

Abstract

Addition of ammonium ions to yeast cells growing on proline as the sole nitrogen source induces rapid inactivation and degradation of the general amino acid permease Gap1 through a process requiring the Npi1/Rsp5 ubiquitin (Ub) ligase. In this study, we show that NH4 + induces endocytosis of Gap1, which is then delivered into the vacuole where it is degraded. This down-regulation is accompanied by increased conversion of Gap1 to ubiquitinated forms. Ubiquitination and subsequent degradation of Gap1 are impaired in thenpi1 strain. In this mutant, the amount of Npi1/Rsp5 Ub ligase is reduced >10-fold compared with wild-type cells. The C-terminal tail of Gap1 contains sequences, including a di-leucine motif, which are required for NH4 +-induced internalization and degradation of the permease. We show here that mutant Gap1 permeases affected in these sequences still bind Ub. Furthermore, we provide evidence that only a small fraction of Gap1 is modified by Ub after addition of NH4 + to mutants defective in endocytosis.

N-Acetylglucosamine-inducible CaGAP1 encodes a general amino acid permease which co-ordinates external nitrogen source response and morphogenesis in Candida albicans

“…In the presence of ammonium, Ure2p, another transcriptional repressor, sterically hinders Gln3p from activating GAP1 (Blinder et al, 1996). These factors bind to an upstream regulatory sequence containing a motif surrounding a core GATA sequence (Springael & Andre, 1998). An obvious similarity between the CaGAP1 promoter and a nitrogenregulated gene promoter like GAP1, GLN1, GDH2, etc., of S. cerevisiae is the presence of a GAATAG sequence (Cunningham & Cooper, 1993).…”

“…Addition of ammonium ions (Springael & Andre, 1998;Bernard & Andre, 2001) or glutamate (Roberg et al, 1997) inhibits the activity of Gap1p in S. cerevisiae. We found in our study that mimosine inhibited the growth of a wildtype strain and transformants (Dgap1 : : CaGAP1) of S. cerevisiae on minimal proline media but was unable to do so in ammonium-or glutamate-containing media.…”

“…The general amino acid permease system is strongly repressed when growth medium contains (NH 4 ) 2 SO 4 and glutamate (Springael & Andre, 1998). To investigate such an effect on the regulation of CaGap1p we did growth kinetics as well as replica plating of wild-type strain MS138, the mutant MS143 (Dgap1) and transformants (Dgap1 : CaGAP1) of S. cerevisiae on media containing ammonia (SAD) and glutamate (SED) as nitrogen source in the presence of mimosine.…”

“…In S. cerevisiae, at least five proteins (Ure2p, Dal80p, Gln3p, Nil1p and Nil2p) function co-ordinately to control the transcription of GAP1 (Blinder et al, 1996;Cunningham et al, 1993;Rowen et al, 1997;Stanbrough et al, 1995). The nitrogendependent regulation of GAP1 is complex, occurring not only at the level of GAP1 transcription but also through Gap1p sorting and degradation by ubiquitin-triggered internalization (Springael et al, 1998).…”

Life in the midst of scarcity: adaptations to nutrient availability in Saccharomyces cerevisiae

“…The protein kinase Npr1 plays a major role in the sorting of these two classes of permeases. Npr1 is required for stabilization of Gap1 at the plasma membrane and induces the degradation of Tat2, possibly by regulating their ubiquitination (Schmidt et al 1998;Springael and Andre 1998;De Craene et al 2001;Helliwell et al 2001;Soetens et al 2001;Springael et al 2002). TORC1 activity, through control of the Tap42-Sit4 phosphatase complex, promotes the phosphorylation of Npr1 (Schmidt et al 1998;Jacinto et al 2001;Gander et al 2008).…”