Amino acid signaling in yeast: casein kinase I and the Ssy5 endoprotease are key determinants of endoproteolytic activation of the membrane-bound Stp1 transcription factor

Abstract

Saccharomyces cerevisiae cells possess a plasma membrane sensor able to detect the presence of extracellular amino acids and then to activate a signaling pathway leading to transcriptional induction of multiple genes, e.g., AGP1, encoding an amino acid permease. This sensing function requires the permease-like Ssy1 and associated Ptr3 and Ssy5 proteins, all essential to activation, by endoproteolytic processing, of the membrane-bound Stp1 transcription factor. The SCF(Grr1) ubiquitin-ligase complex is also essential to AGP1 induction, but its exact role in the amino acid signaling pathway remains unclear. Here we show that Stp1 undergoes casein kinase I-dependent phosphorylation. In the yck mutant lacking this kinase, Stp1 is not cleaved and AGP1 is not induced in response to amino acids. Furthermore, we provide evidence that Ssy5 is the endoprotease responsible for Stp1 processing. Ssy5 is significantly similar to serine proteases, its self-processing is a prerequisite for Stp1 cleavage, and its overexpression causes inducer-independent Stp1 cleavage and high-level AGP1 transcription. We further show that Stp1 processing also requires the SCF(Grr1) complex but is insensitive to proteasome inhibition. However, Stp1 processing does not require SCF(Grr1), Ssy1, or Ptr3 when Ssy5 is overproduced. Finally, we describe the properties of a particular ptr3 mutant that suggest that Ptr3 acts with Ssy1 in amino acid detection and signal initiation. We propose that Ssy1 and Ptr3 form the core components of the amino acid sensor. Upon detection of external amino acids, Ssy1-Ptr3 likely allows-in a manner dependent on SCF(Grr1)-the Ssy5 endoprotease to gain access to and to cleave Stp1, this requiring prior phosphorylation of Stp1 by casein kinase I.

FIG. 1.

CKI is essential to AGP1 induction in response to amino acids. The wild type (LRB341) and the yck1Δ-1 yck2-2^ts (LRB346, yck^ts) strain were transformed with the YCpAGP1-lacZ plasmid (URA3) and the YCpJYS-20 plasmid bearing the HIS3 and LEU2 genes for complementation of auxotrophies. The strains were grown at 24°C on minimal medium containing urea as sole nitrogen source. Half of the cultures were transferred to 37°C for 20 min. Phenylalanine was then added (+) or not (−), and the cultures were incubated for 2 h. Then, β-galactosidase activity was measured as described in Materials and Methods. Total cellular extracts were also analyzed in a Western blotting experiment using anti-Agp1 antibodies.

FIG. 2.

Role of CKI in phosphorylation and endoproteolytic processing of Stp1. The wild-type (LRB341) and yck1Δ yck2-2^ts (LRB346, yck^ts) strains were transformed with plasmid pCA047 (URA3) bearing the STP1-HA gene and plasmid YCpJYS-20 bearing the HIS3 and LEU2 genes for complementation of auxotrophies. (A) The strains were grown at 24°C on minimal medium containing urea as sole nitrogen source. Cultures were then transferred to 37°C for 20 min. Phenylalanine was then added (+) or not (−) to the cultures, which were then incubated for 2 h before immunoblot analysis of total cell extracts with anti-HA antibodies. (B) The strains were grown at 24°C on urea medium, and total cell extracts were analyzed by immunoblotting with anti-HA antibodies. (C) The strains were grown at 24°C on urea medium. Cultures were then transferred to 37°C for 20 min, and total cell extracts were incubated (+) or not (−) in the presence of alkaline phosphatase (Pse) for 1 h. After migration, immunoblotting was carried out with anti-HA antibodies.

FIG. 3.

Components of the SCF^Grr1 ubiquitin-ligase complex are essential to amino acid-induced endoproteolytic processing of Stp1. (A) Wild-type (23344c) and grr1Δ (JA115) strains transformed with the pCA047 plasmid (STP1-HA) were grown on minimal medium with proline as sole nitrogen source. Phenylalanine (5 mM final concentration) was added (+) or not (−), and the culture was incubated for 20 min. Crude cell extracts were then prepared, and immunoblotting was carried out with anti-HA antibodies. (B) Wild-type (W303), cdc34-1 (MT670), cdc4-1 (MT668), and grr1Δ (JA115) strains transformed with the pCA047 plasmid (STP1-HA) were grown at 30°C on minimal medium with proline as sole nitrogen source. Cells were transferred to 37°C and incubated for 60 min. Then, phenylalanine (5 mM final concentration) was added (+) or not (−), and the cultures were incubated for an additional 15 min. Crude cell extracts were then prepared, and immunoblotting was carried out with anti-HA antibodies.

FIG. 4.

Stp1 cleavage in response to amino acids is insensitive to proteasome inhibition. (A) Strain pdr5Δ (JA547) transformed with plasmid pCA047 (STP1-HA) was grown on minimal proline medium. The proteasome inhibitor MG132 (50 μM final concentration) was added (or not), and the cultures were incubated for 2 h before addition of phenylalanine (5 mM). Before and several times after addition of the amino acid, culture samples were collected and crude extracts were analyzed by immunoblotting with anti-HA antibodies. (B) The same extracts of cells grown without MG132 (−) or incubated with MG132 (+) for 90 min (lane 2) or 120 min (lane 3) were analyzed by immunoblotting with antibodies against Gic2.

FIG. 5.

Overexpression of SSY5 suppresses the growth defects caused by the grr1Δ, ssy1Δ, and ptr3Δ mutations. Cells were spread on minimal medium with leucine (1 mM) as sole nitrogen source or on minimal NH₄⁺ medium containing the toxic amino acid analogue l-ethionine (20 μg · ml⁻¹). Glucose (Glu) or galactose (Gal) was added as sole carbon source. The strains were FA38 (GAL1-HA-SSY5 gap1Δ ura3), FA39 (GAL1-HA-SSY5 gap1Δ ssy1Δ ura3), FA20 (GAL1-HA-SSY5 gap1Δ ptr3Δ ura3), 38016c (GAL1-HA-SSY5 gap1Δ grr1Δ ura3), 30629c (gap1Δ ura3), 34065b (gap1Δ ssy1Δ ura3), FB94 (gap1Δ ptr3Δ ura3), and FA08 (gap1Δ grr1Δ ura3).

FIG. 6.

Overproduction of Ssy5 is accompanied by its endoproteolytic processing and causes constitutive Stp1 cleavage and Agp1 synthesis. (A) Strains FA38 (GAL-HA-SSY5, noted as wild type) and 38016c (GAL-HA-SSY5 grr1Δ, noted as grr1Δ) were grown on raffinose medium with proline as the sole nitrogen source. At time zero, galactose was added to the medium. Crude extracts were prepared from culture samples collected before and at the indicated time intervals after galactose addition, and immunoblotting was carried out with either anti-HA (upper panel) or anti-Agp1 (lower panel) antibodies. (B) Strain 38003a (GAL1-HA-SSY5) transformed with the CEN-based plasmid pCA047 (STP1-HA) was grown on minimal raffinose medium with proline as the sole nitrogen source. Crude extracts were prepared from culture samples collected before (time zero) and at two time intervals after galactose addition, and immunoblotting was carried out with anti-HA antibodies. The immunodetected signals corresponding to the full-length (Ssy5, Stp1) and processed (Ssy5*, Stp1*) forms of Ssy5 and Stp1 are indicated. (C) Strains FA44 (GAL1-HA-SSY5, noted as wild type) and FA101 (yck1Δ1 yck2-2^ts GAL1-HA-SSY5, noted as yck^ts), both transformed with the YCpJYS-20 plasmid to complement auxotrophies and the pCA047 plasmid (STP1-HA), were grown at 24°C on raffinose medium with proline as the sole nitrogen source. Cultures were then transferred to 37°C for 20 min. At time zero, galactose was added to the medium. Crude extracts were prepared from culture samples collected before and at the indicated times after galactose addition and immunoblotted using anti-HA antibodies. (D) Strain 34692c (ssy5Δ) transformed with the pFA150 plasmid (HA-SSY5) was grown on minimal medium with proline as sole nitrogen source. Phenylalanine (5 mM final concentration) was added (+) or not (−), and the culture was incubated for 30 min. Crude cell extracts were then prepared, and immunoblotting was carried out with anti-HA antibodies.

FIG. 7.

The C-terminal region of Ssy5 shows similarity to S1-family serine proteases. (Top) Schematic representation of the domain structure of the Ssy5 protein. The C-terminal serine-protease-like domain (residues 459 to 687) defined by the Superfamily program is boxed. The approximate relative positions of the histidine (H), aspartate (D), and serine (S) residues corresponding to the probable catalytic endoprotease site of Ssy5 are indicated. The black box just upstream from the protease domain represents the position of the putative cleavage site of Ssy5 (see Fig. 8). The horizontal line with arrows delimits the Ssy5 region most highly conserved between Ssy5 proteins of various fungal species. (Bottom) Sequence logos taken from HMM 0011293 around residues of the catalytic triad (see text). The greater the height of the amino acid (single-letter code), the higher its conservation in the HMM alignment. Ssy5 sequences around the three amino acids (positions 465, 545, and 640) are also shown. The seed sequence used to initiate building of HMM 0011293 was the catalytic domain of the HtrA protease of E. coli (39). The sequence of the catalytic triad of HtrA is also shown.

FIG. 8.

Ssy5 self-processing is required for Stp1 cleavage and Agp1 synthesis. (A) A serine-to-alanine substitution in the predicted catalytic site of Ssy5 impairs amino acid-induced Stp1 cleavage and Agp1 synthesis. Strain 34686b (ssy5Δ, STP1-HA) transformed with the CEN-based plasmid pFA153 (SSY5) or pFA154 (SSY5^S→A) was grown on minimal medium with proline as sole nitrogen source. Phenylalanine (5 mM final concentration) was added (+) or not (−), and the culture was incubated for 45 min. Crude cell extracts were then prepared, and immunoblotting was carried out with anti-HA (upper panel) or anti-Agp1 (lower panel) antibodies. (B) A serine-to-alanine substitution in the predicted catalytic site of Ssy5 impairs Ssy5 processing, Stp1 cleavage, and Agp1 synthesis under conditions of Ssy5 overproduction. Strain 34686b (ssy5Δ STP1-HA) transformed with the CEN-based plasmid pFA138 (GAL1-HA-SSY5) or pFA144 (GAL1-HA-SSY5^S→A) was grown on minimal raffinose medium with proline as the sole nitrogen source. At time zero, galactose was added to the medium. Crude extracts were prepared from culture samples collected before and 120 min after galactose addition, and immunoblotting was carried out with either anti-HA (upper panel) or anti-Agp1 (lower panel) antibodies. (C) Conservation of a putative endoproteolytic cleavage site in the Ssy5 and Stp1 proteins. The alignment was generated with CLUSTAL and the following Ssy5-orthologous sequences: Sc-Ssy5, Sc-Stp1, and Sc-Stp2 (S. cerevisiae, Ssy5 positions 409 to 432, Stp1 positions 84 to 107, Stp2 positions 92 to 115), Sp-Ssy5 and Sp-Stp1 (Saccharomyces paradoxus), Sm-Ssy5 and Sm-Stp1 (Saccharomyces mikatae), Sb-Ssy5 andSb-Stp1 (Saccharomyces bayanus), Sca-Ssy5 and Sca-Stp1 (Saccharomyces castellii), Eg-Ssy5 and Eg-Stp1 (Eremothecium gossypii), Kw-Ssy5 and Kw-Stp1 (Kluyveromyces waltii), Sk-Ssy5 and Sk-Stp1 (Saccharomyces kluyveri), Yl-Ssy5 (Yarrowia lipolytica), and Ca-Ssy5 (Candida albicans). All sequences were retrieved from the SGD database (http://www.yeastgenome.org/). (D) A 4-amino-acid deletion in the putative endoproteolytic cleavage site of Ssy5 impairs Ssy5 self-processing and Stp1 cleavage. Strain 34686b (ssy5Δ STP1-HA) transformed with the CEN-based plasmid pFA138 (GAL1-HA-SSY5) or pFA148 (GAL1-HA-SSY5^PISMSLΔ) was grown on minimal raffinose medium with proline as the sole nitrogen source. At time zero, galactose was added to the medium. Crude extracts were prepared from culture samples collected before and 120 min after galactose addition, and immunoblotting was carried out with anti-HA antibodies.

FIG. 9.

The ptr3-35 mutant responds to external leucine but not to other amino acids. Cells were spread on minimal medium with the indicated amino acid (1 mM) as sole nitrogen source. The strains were 30629c (gap1Δ ura3), FB94 (gap1Δ ptr3Δ ura3), FB35 (gap1Δ ptr3-35 ura3), and strain FB94 transformed by the CEN-based plasmid YCp-ptr3-35. Growth on these media is indicative of the expression of an active Agp1 permease (31).