Regulation of transcription factor latency by receptor-activated proteolysis

Abstract

The transcription factor Stp1 is endoproteolytically processed in response to extracellular amino acids by the plasma membrane SPS (Ssy1-Ptr3-Ssy5)-sensor. Processed Stp1, lacking a cytoplasmic retention motif, enters the nucleus and induces amino acid transporter gene expression. The SPS-sensor component Ssy5 is a chymotrypsin-like protease with a Pro-domain and a catalytic domain. The Pro-domain, required for protease maturation, is autolytically cleaved from the catalytic domain but remains associated, forming an inactive protease complex that binds Stp1. Stp1 is processed only after amino acid-induced signals cause the dissociation of the inhibitory Pro-domain. Our findings demonstrate that gene expression can be controlled by regulating the enzymatic activity of an intracellular endoprotease.

Figure 1.

The N-terminal domain of Stp1 interacts with Ssy5. (A) Schematic representation of Stp1; the location of the regulatory domain (REG) and DNA-binding domains (DBD) are depicted in the full-length Stp1 (519 amino acids) (Andréasson and Ljungdahl 2002). Conserved sequence motifs, Region I (amino acids 16–35) and Region II (amino acids 65–97), are indicated within the enlargement of the N-terminal domain (amino acids 1–125) (Andréasson and Ljungdahl 2004). A two-hybrid screen using the LexA–Stp1_(1–125) fusion protein as bait identified Ssy5 as a potential Stp1-processing protease. The Stp1 interaction motif was mapped using the diagrammed bait constructs. (B) Residues 63–125 of Stp1 suffice for amino acid-induced Ssy5-dependent processing. Immunoblotting of cell extracts from strain CAY309 [pRS317] carrying PGAL1-ZZ-STP1_63–125-GST (pCA221), and either pRS316 (ssy5Δ) or pFL001 (SSY5) grown in SGal medium and harvested 30 min after induction by leucine as indicated. The immunoreactive forms of ZZ-STP1_63–125-GST are schematically depicted at their corresponding positions of migration. (C) Ssy5 physically associates with the N-terminal domain of Stp1. (Lanes 2–5) Beads with immobilized ZZ-myc or ZZ-Stp1_63–125-myc proteins as indicated were incubated with one volume (+) or two volumes (++) of Ssy5-GST-containing lysates and washed extensively, and bound material was eluted in sample buffer. Samples were resolved by SDS-PAGE together with 5% of input lysate (cf. Ssy5-GST ++, lane 1), and an aliquot of extract after incubation with beads (FT, lanes 6,7). The immunoreactive forms of Ssy5-GST present in the cell extracts are schematically shown at their corresponding positions of migration.

Figure 2.

The Pro-domain of Ssy5 is essential for protease maturation and activity. Schematic representation of the domain structure of Ssy5 (699 amino acids). The Pro-domain (amino acids 1–381) and Cat-domain (amino acids 382–699) are depicted. The endoproteolytic-cleavage site is indicated (scissors) (Poulsen et al. 2006). Dilutions of cultures of strain CAY328 (ssy5Δ) carrying plasmids as indicated—i.e., empty vectors pRS316 and pRS314 (control), pCA174 (Ssy5-GST), pSH076 (HA-Pro-ssy5), pSH087 (Cat-ssy5-GST), or both pSH076 and pSH087, respectively, grown in SD—were spotted onto YPD and YPD supplemented with MM (YPD + MM). MM, a sulfonylurea analog, inhibits branched chained amino acid synthesis (Jørgensen et al. 1998). Plates were incubated for 2 d at 30°C. Immunoblot analysis of extracts from strains; the immunoreactive forms of epitope-tagged proteins are schematically represented at their corresponding positions of migration. Stars mark the position of unrelated cross-reacting proteins.

Figure 3.

Pro-domain of Ssy5 functions to inhibit Stp1 processing in the absence of amino acid-induced signaling. (A) Stp1 is proteolytically processed when coexpressed with HA-Ssy5 in S. pombe. Immunoblot analysis of extracts from S. cerevisiae ssy5Δ strain HKY77 [pCA204 (Stp1-myc)] carrying either pCA213 (SSY5-Flag) or pCA214 (HA-SSY5-Flag) grown in SD (−leu) and 30 min after the induction by leucine (+leu) (lanes 1–4), and from S. pombe strains CAY301 (control), CAY326 (SSY5-Flag), and CAY327 (HA-SSY5-Flag) grown in YES. Stars mark the position of unrelated cross-reacting proteins. (B) In vitro processing of recombinant Stp1 in cell-free lysates. Lysates were prepared from noninduced (−leu) and leucine-induced (+leu) cultures of wild-type (WT, CAY29), ssy1Δ (CAY91), ptr3Δ (JAY7), ssy5Δ (JAY14), grr1Δ (CAY86), and stp1Δ stp2Δ (CAY123) strains grown in uracil-supplemented SD. Recombinant Stp1-HA was added to the lysates; after 1 h the reactions were resolved by SDS-PAGE and analyzed by immunoblotting.

Figure 4.

The Pro- and Cat-domains copurify, and amino acid-induced signals reduce Pro-domain levels. (A) GST fusion proteins were affinity-purified from extracts from SD-grown CAY324 (ssy5Δ prb1Δ) carrying pCA229 (Ssy5-GST) or pCA230 (HA-Ssy5-GST). Bound protein was eluted, resolved by SDS-PAGE, and either stained with Coomassie (lanes 1,2) or immunoblotted with α-GST (lanes 3,4) or α-HA (lanes 5,6) antibodies. (B) Time course of leucine-induced Stp1 processing. SD-grown cultures of ssy5Δ strain HKY77 (pCA204 [Stp1-myc]) carrying pFL005 (HA-Ssy5, top panel) or pCA177 (Ssy5-HA, middle and bottom panels) were induced with leucine. Immunoblot analysis of extracts from subsamples of each culture were removed at 5-min intervals after leucine addition. The signal intensity (arbitrary units) of the immunoreactive band corresponding to the HA-tagged Pro-domain was quantified; the values normalized to the t = 0 time point are plotted. (C) Immunoblot analysis of extracts from ssy1Δ (HKY84), ptr3Δ (HKY85), and grr1Δ (CAY274) strains carrying pFL005 (top panel) or pCA177 (bottom panel) grown in SD and 30 min after cells were induced with leucine. The immunoreactive forms of epitope-tagged proteins are schematically depicted at their corresponding positions of migration. Stars mark the position of unrelated cross-reacting proteins.

Figure 5.

Model of the experimentally determined two-step activation of the Ssy5 protease. Ssy5 is expressed as a full-length and inactive zymogen (pro-Ssy5) that is autolytically processed (scissors), and thus catalytically competent, but inhibited by its interaction with the Pro-domain (primed). The plasma membrane receptor Ssy1, together with Ptr3, transduces amino acid-initiated signals, resulting in the release of Pro-domain inhibition. Stp1 and Stp2 are processed (scissors) by the active Cat-domain. The processed forms of Stp1 and Stp2, lacking N-terminal negative regulatory domains, enter the nucleus, bind relevant promoters, and activate gene expression.