Yeast Gcn4p stabilization is initiated by the dissociation of the nuclear Pho85p/Pcl5p complex

Abstract

Protein stability of the c-jun-like yeast bZIP transcriptional activator Gcn4p is exclusively controlled in the yeast nucleus. Phosphorylation by the nuclear Pho85p cyclin-dependent protein kinase, a functional homolog of mammalian Cdk5, initiates the Gcn4p degradation pathway in complex with the cyclin Pcl5p. We show that the initial step in Gcn4p stabilization is the dissociation of the Pho85p/Pcl5p complex. Pcl7p, another nuclear and constantly present cyclin, is required for Gcn4p stabilization and is able to associate to Pho85p independently of the activity of the Gcn4p degradation pathway. In addition, the nuclear cyclin-dependent Pho85p kinase inhibitor Pho81p is required for Gcn4p stabilization. Pho81p only interacts with Pcl5p when Gcn4p is rapidly degraded but constitutively interacts with Pcl7p. Our data suggest that Pcl7p and Pho81p are antagonists of the Pho85p/Pcl5p complex formation in a yet unknown way, which are specifically required for Gcn4p stabilization. We suggest that dissociation of the Pho85p/Pcl5p complex as initial step in Gcn4p stabilization is a prerequisite for a shift of equilibrium to an increased amount of the Pho85p/Pcl7p complexes and subsequently results in decreased Gcn4p phosphorylation and therefore increased stability of the transcription factor.

Figure 1.

A pho81 mutation leads to unstable Gcn4p in amino acid–starved yeast cells. (A) The isogenic S. cerevisiae strains RH2711 (PHO81) and RH2712 (pho81) were transformed to express the GAL1-driven myc³-GCN4 on plasmid KB294. (B) The pho81 mutant strains RH3306 and RH3307 express endogenous GCN4-myc⁹ from the authentic GCN4 promoter under glucose conditions or from the GALL promoter in galactose containing medium. (C) In addition, GCN4 was expressed from the GAL1 promoter (KB294) together with MET25 driven PHO81 (pME2863) in the pho81 mutant strain RH3241 (pho81/Pho81p). Protein levels of myc³-Gcn4p, Gcn4-myc⁹p and Cdc28p or eIF2p as loading control were determined in sated (+Leu) and amino acid–starved (−Leu) cells after the GAL promoter shut-off. A twofold protein amount was loaded for overexpressed PHO81 to obtain similar amounts of Gcn4p at time point 0. Numbers given below indicate remaining Gcn4p percentages when compared with Cdc28p or eIF2p as internal standard quantified by image station of the gel shown.

Figure 2.

Nuclear localization and stability of Pho81p are unaffected by the availability of amino acids. (A) Nuclear import of Pho81p is independent of the presence or absence of amino acids. Yeast pho81 mutant strain RH2712 was transformed to express GFP-PHO81 driven from the MET25 promoter in high amounts (pME2228). Cells were analyzed under sated (+Leu) and starved (−Leu) conditions. On the left, DIC microscopy is shown and on the right, GFP and DAPI signals are merged (Merge). (B) Amino acid starvation results in unchanged GFP-Pho81p protein stability. The pho81-deficient yeast strain RH2712 was transformed to express GFP-PHO81 from the repressible MET25 promoter on a 2-μm plasmid (pME2228). Protein levels of GFP-Pho81p and Cdc28p as loading controls were determined in sated (+Leu) and amino acid–starved (−Leu) cells after the MET25 promoter shut-off.

Figure 3.

Protein–protein interaction of Pcl5p with Pho81p and Pho85p are disrupted in S. cerevisiae when Gcn4p is stabilized. The strain RH3241 (pho81) (A) was transformed to express either myc⁹-PCL5 (pME2865) with glutathione S transferase (GST on pYGEX-2T) as negative control (NC), or myc⁹-PCL5 (pME2865) together with GST-PHO81 (pME2867). In addition, yeast strains RH3238 (PHO81) (B) and RH3241 (pho81) (C) were transformed to express either myc⁹-PCL5 (pME2865) with glutathione S-transferase (GST on pYGEX-2T) as negative control, or myc⁹-PCL5 (pME2865) together with GST-PHO85 (pME2866). Protein levels of the fusion proteins were determined in sated (+aa) and amino acid–starved (−aa) cells. The left part represents the GST, GST-Pho81p, GST-Pho85p, and myc⁹-Pcl5p before GST-agarose incubation to ensure that the initial protein extracts (PE) contain similar amounts of the fusion proteins. On the right, the elutions of the glutathione beads are shown (Beads). The twofold amount of protein extract and elution of the glutathione beads of amino acid–starved cells were loaded in case of the myc-antibody reaction in the pho81 mutant strain (A and C) to obtain similar amounts of myc⁹-Pcl5p in the initial protein extract (A).

Figure 4.

A pcl7 mutation leads to unstable Gcn4p in amino acid–starved yeast cells. (A) The isogenic yeast strains RH3237 (PCL7) and RH3255 (pcl7) were transformed to express myc³-GCN4 from the GAL1 promoter from the high copy number plasmid KB294. (B) In addition GCN4 was expressed from the GAL1 promoter (KB294) together with MET25 driven PCL7 (pME2933) in the pcl7 mutant strain RH3255 (pcl7/Pcl7p). Protein levels of myc³-Gcn4p and Cdc28p or eIF2p as loading control were determined in sated (+Leu) and amino acid–starved (−Leu) cells after the GAL1 promoter shut-off. In addition, phosphorylated eIF2α-Pp confirms amino acid starvation. Numbers given below indicate remaining Gcn4p-percentages when compared with eIF2p as internal standard quantified by image station of the gel shown. (C) Overexpression of PCL7 results in sensitivity toward amino acid starvation. Yeast strains RH3237 (PCL7), RH3255 (pcl7), and pcl7 mutant cells expressing PCL7 (pME2933) from the MET25 promoter on 2-μm plasmids (pcl7/Pcl7p) were spotted in fivefold dilution on minimal medium (YNB), YNB with 100 mM 3AT, and YNB with 100 mM 3AT and histidine. Plates were incubated at 30°C for 3 d.

Figure 5.

Nuclear localization and stability of cyclin Pcl7p are unaffected by the availability of amino acids. (A) The GFP-Pcl7p protein fusion is predominantly enriched in the yeast nucleus in sated and amino acid–starved cells. pcl7 mutant cells (RH3255) expressing GFP-PCL7 from the MET25 promoter (pME2230) were analyzed under sated (+Leu) and starved (−Leu) conditions by DIC microscopy and fluorescence microscopy (GFP). (B) GFP-Pcl7p is a stable yeast protein independently of the availability of amino acids. The yeast strain RH1168 was transformed to express GFP-PCL7 (pME2230). Protein levels of GFP-Pcl7p and Cdc28p as loading control were determined in sated (+Leu) and amino acid–starved (−Leu) cells after the MET25 promoter shut-off. The GFP antibody recognizes additional bands of different sizes, which might be products of premature translation termination or protein degradation.

Figure 6.

Pcl7p interacts with Pho81p and Pho85p independently of the stability of Gcn4p. The S. cerevisiae strain RH2977 containing PCL7-myc⁹ behind its endogenous promoter expresses either PCL7-myc⁹ with glutathione S-transferase (GST on pYGEX-2T) as negative control (NC), PCL7-myc⁹ together with GST-PHO81 (pME2867) (A) or PCL7-myc⁹ together with GST-PHO85 (pME2866) (B). In addition, yeast strain RH3255 (pcl7) was transformed to express either myc⁹-PCL5 (pME2865) with glutathione S-transferase (GST on pYGEX-2T) as negative control, or myc⁹-PCL5 (pME2865) together with GST-PHO85 (pME2866) (C). Protein levels of the fusion proteins were determined in sated (+aa) and amino acid–starved (−aa) cells. Left, GST, GST-Pho81p, GST-Pho85p and Pcl7-myc⁹p before GST-agarose incubation (PE). Right, the elutions of the glutathione beads (Beads).

Figure 7.

Model for Gcn4p stability regulation. In sated cells, when amino acids are available (+Amino Acids), both cyclins, Pcl5p, and Pcl7p compete for binding to the kinase Pho85p. This results in a possible equilibrium of both complexes with sufficient amounts of Pho85p/Pcl5p for Gcn4p phosphorylation and therefore rapid protein degradation. In response to amino acid starvation (−Amino Acids), Gcn4p is stabilized because the Pho85p/Pcl5p complex is dissociated, and Pcl5p is replaced by Pcl7p, which is present in high amounts. The decrease of Pho85p/Pcl5p complexes results in less phosphorylation of Gcn4p and subsequently in its stabilization.