The N-terminal regulatory domain of Stp1p is modular and, fused to an artificial transcription factor, confers full Ssy1p-Ptr3p-Ssy5p sensor control

Abstract

Stp1p and Stp2p are homologous and redundant transcription factors that are synthesized as latent cytoplasmic proteins with N-terminal regulatory domains. In response to extracellular amino acids, the plasma membrane-localized Ssy1p-Ptr3p-Ssy5p (SPS) sensor induces an endoproteolytic processing event that cleaves away the N-terminal regulatory domains. The shorter forms of Stp1p and Stp2p are targeted to the nucleus, where they bind and activate the transcription of amino acid permease genes. A novel genetic screen, specifically designed to search for rare mutations that affect the SPS-sensing pathway, identified the F-box protein Grr1p as an obligatory factor required for Stp1p/Stp2p processing. Additionally, we have found that a null mutation in the ASI1 (amino acid sensor-independent) gene enables full-length unprocessed Stp1p/Stp2p to enter the nucleus and derepress SPS sensor-dependent genes. The N-terminal domains of Stp1p/Stp2p contain two conserved motifs that are required for proper nuclear exclusion and proteolytic processing. These motifs function in parallel; mutations that abolish processing inhibit signaling, whereas mutations that interfere with cytoplasmic retention result in constitutive derepression of SPS sensor-regulated genes independently of processing. The N-terminal domain of Stp1p is functionally autonomous and transferable to other transcription factors, where its presence confers ASI1-dependent nuclear exclusion and SPS sensor-induced proteolytic processing.

FIG. 1.

The N-terminal regulatory domains of the Stp1p and Stp2p orthologues contain two distinct regions of sequence conservation. (A) Schematic diagram of the SPS sensor pathway. Extracellular amino acids activate the SPS sensor, induce the endoproteolytic processing of transcription factors Stp1p and Stp2p, and thereby induce the expression of multiple amino acid permease genes. Consequently, cells grown in the presence of extracellular amino acids exhibit robust amino acid uptake and are sensitive to toxic amino acid analogues, e.g., AzC. (B) The first 125 amino acid residues from Stp1p/Stp2p (S.c.) and their orthologues in S. bayanus (S.b.), S. mikatae (S.m.), and S. paradoxus (S.p.) were aligned by using the CLUSTAL W algorithm (37). Conserved (asterisks) and similar (colons and periods) residues are marked. Regions I and II (gray boxes) are defined by stretches of enhanced sequence conservation. (C) Schematic representation of Stp1p. The locations of the inhibitory domain (REG), defined by the breakpoint of the STP1Δ133 allele (1), the DNA-binding domains (DBD), and the putative NLS are depicted in the full-length Stp1p (519 amino acids). Region I (amino acids 16 to 35), region II (amino acids 65 to 97), and the alanine substitution mutations of the STP1-133 and stp1-102 alleles are indicated in the enlargement of the N-terminal domain (amino acids 1 to 125).

FIG. 2.

Mutations in regions I and II of Stp1p demonstrate the modular nature of the N-terminal inhibitory domain. (A) The stp1-102 allele encodes a mutant protein that is not endoproteolytically processed. (Left panel) Immunoblot analysis of protein extracts from strain CAY123 (stp1Δ stp2Δ) transformed with plasmids carrying wild-type STP1 (pCA047) or mutant allele L65→A (pCA127), F66→A (pCA129), or P67→A (pCA128). Cells were grown in SD medium (−leu) and, where indicated, leucine was added 30 min prior to harvest (+leu). Extracts were resolved by SDS-PAGE and immunoblotted with anti-HA. The immunoreactive forms of Stp1p present in the cell extracts are schematically represented at their corresponding positions of migration. (Right panel) Tenfold dilution series of cell suspensions of strain CAY123 (stp1Δ stp2Δ) transformed with pRS316 (Vector), pCA047 (STP1), pCA127 (L65→A), pCA129 (F66→A), or pCA128 (P67→A) were spotted on plates containing SD medium (top) and SD medium supplemented with leucine and AzC (bottom). (B) The STP1-133 allele encodes a constitutively active protein. (Left panel) Tenfold dilution series of cell suspensions of an ssy1Δ strain (HKY20) cotransformed with pCA030 (PAGP1-lacZ) and either pRS316 (Vector), pCA047 (STP1), pCA027 (STP1Δ131), or pCA120 (STP1-133) were spotted on plates containing SD medium (top) and SD medium supplemented with leucine and AzC (bottom). (Middle panel) Immunoblot analysis of protein extracts from HKY20 transformants grown in SD medium. Extracts were resolved by SDS-PAGE and immunoblotted with anti-HA. (Right panel) β-Galactosidase activity (Miller units, with standard deviations [n = 4]) present in each strain was quantified to monitor the expression of the PAGP1-lacZ reporter construct. (C) The STP1-133 mutation and the asi1Δ allele can suppress noncleavable stp1-102. (Left panel) Immunoblot analysis of protein extracts from strains CAY152 (asi1Δ) and CAY123 (ASI1) transformed with a plasmid carrying wild-type STP1 (pCA047) or mutant allele stp1-102 (pCA129), STP1-133 (pCA120), or STP1-102,133 (pCA135). Cells were grown in SD medium, and leucine was added 30 min priorto harvest. Extracts were resolved by SDS-PAGE and immunoblotted with anti-HA. (Right panel) Tenfold dilution series of cell suspensions of strains CAY152 (stp1Δ stp2Δ asi1Δ) and CAY123 (stp1Δ stp2Δ ASI1) transformed with pRS316 (Vector), pCA047 (STP1), pCA129 (stp1-102), pCA120 (STP1-133), or pCA135 (STP1-102,133) were spotted on plates containing SD medium (top) and SD medium supplemented with leucine and AzC (bottom).

FIG. 3.

A genetic screen identifies GRR1 as an obligate component of the amino acid-induced SPS sensor pathway. (A) Merodiploid strains carrying duplicated genes encoding the known SPS sensor components and SHR3 were constructed by integrating PTR3 and SSY1 into the ADE2 locus on chromosome (Chr) XV and SSY5 and SHR3 into the CAN1 locus on chromosome V, creating the ade2Δ::PTR3-loxP-kanMX-loxP-SSY1 and can1Δ::SSY5-natMX-SHR3 alleles, respectively. (B) Tenfold dilution series of strain CAY241 (wild type [WT]) and six spontaneous AzC-resistant mutants lacking detectable β-galactosidase activity were applied as drops to solid SD medium (top) and SD medium containing leucine and AzC (bottom). The plates were incubated at 30°C for 7 days and photographed. β-Galactosidase activity (Miller units) present in each strain was quantified to monitor the expression of the integrated PAGP1-lacZ reporter construct. (C) Complementation analysis of diploid strains generated by mating CAY241 (WT) and AzC-resistant LacZ-negative mutants (mutants 1 to 6) with PLY126 (WT) and CAY103 (grr1Δ). The diploid strains were patched on SD medium supplemented with leucine, uracil, and AzC (top) and on SC medium (bottom). The plates were incubated at 30°C for 2 days, and β-galactosidase activity was visualized on SC medium by using an X-Gal overlay. (D) Tenfold dilution series of cell suspensions of mutant 1 (Mut 1), a WT strain (CAY241), or a grr1Δ strain (CAY86) transformed with pRS316 (−) or pCA212 (+) were spotted on plates containing SD medium (top) and SD medium supplemented with leucine and AzC (bottom). The plates were incubated at 30°C for 4 days and photographed.

FIG. 4.

Loss-of-function mutations in GRR1 inhibit the endoproteolytic processing of Stp1p. (A) Immunoblot analysis of whole-cell extracts from a wild-type (WT) strain (CAY241) and AzC-resistant LacZ-negative mutant strains (Fig. 3) transformed with pCA171 (STP1-HA). Cells were grown in SD medium supplemented with adenine and uracil. Leucine (leu) was added 30 min prior to harvest. (B) Immunoblot analysis of whole-cell extracts from a WT strain (CAY29), an ssy1Δ strain (CAY91), and a grr1Δ strain (CAY86) transformed with pCA047 (STP1-HA). Cells were grown in SD medium. Leucine (leu) was added 30 min prior to harvest.

FIG. 5.

Epistasis analysis of grr1Δ and SPS-sensing pathway mutations. (A) Immunoblot analysis of whole-cell extracts from a wild-type (WT) strain (PLY126) and a grr1Δ strain (CAY103) cotransformed with pCA122 (STP1-HA) and either pHK010 (SSY1) or pSSY1-102 (SSY1-102). Cells were grown in SD medium, and leucine (leu) was added 30 min prior to harvest. (B) Tenfold dilution series of cell suspensions of WT (CAY29), ssy1Δ (CAY91), grr1Δ (CAY86), asi1Δ (CAY144), ssy1Δ asi1Δ (CAY206), and grr1Δ asi1Δ (CAY109) strains carrying plasmid pRS316 and STP1Δ131 (CAY29), ssy1Δ STP1Δ131 (CAY91), and grr1Δ STP1Δ131 (CAY86) strains carrying plasmid pCA027 were spotted on plates containing SD medium (top) and SD medium supplemented with AzC (bottom). The plates were incubated at 30°C.

FIG. 6.

The N-terminal regulatory domain of Stp1p is transferable and confers full SPS sensor control of an artificial transcription factor. (A) Immunoblot analysis of protein extracts from wild-type (WT) (PLY126), ssy1Δ (HKY20), ptr3Δ (HKY31), ssy5Δ (HKY77), grr1Δ (CAY103), and asi1Δ (CAY107) strains transformed with pCA161 [Stp1_(1-125)-LexA-AD] and grown in SD medium with or without leucine (leu). Extracts were resolved by SDS-PAGE and immunoblotted with anti-LexA antibodies. (B) Strains from panel A carrying pSH18-34 (Op_lexA-lacZ) and either pCA160 (vector) or pCA161 [Stp1_(1-125)-LexA-AD] were grown in SD medium with or without leucine (leu). The levels of X-Gal staining resulting from the expression of lexA operator-promoted β-galactosidase in permeabilized cells were assessed.

FIG. 7.

Model of the amino acid-induced SPS-sensing pathway. The latent nature of Stp1p/Stp2p is determined by conserved sequence motifs region I (red) and region II (green) present in their N-terminal domains (orange). Region I appears to function as a cytoplasmic retention signal (anchor) that prevents the unprocessed full-length forms from efficiently entering the nucleus. Region II is required for amino acid-induced endoproteolytic processing (scissor). In the absence of Asi1p, full-length Stp1p/Stp2p can enter the nucleus at rates sufficient to partially derepress amino acid permease (AAP) gene expression. Extracellular amino acids induce the proteolytic processing of Stp1p/Stp2p in an SPS sensor- and Grr1p-dependent manner. The precise function of Grr1p remains to be defined (?); Grr1p either functions concomitantly with the previously characterized SPS sensor components (Ssy1p-Ptr3p-Ssy5p) or is required for SPS sensor assembly. The processed shorter forms of Stp1p and Stp2p containing DNA-binding domains (white boxes) accumulate in the nucleus, where they bind UAS present within the promoters of amino acid permease genes (UAS_aa). PM, plasma membrane; NM, nuclear membrane.