![[Feedback]](/icons/banner/feedbackACT.gif){kind=icon}
![[For Subscribers]](/icons/banner/subscriberACT.gif){kind=icon}
![[Archive]](/icons/banner/archiveACT.gif){kind=icon}
![[Search]](/icons/banner/searchACT.gif){kind=icon}
![[Contents]](/icons/banner/tocACT.gif){kind=icon}
Previous Article | Next Article 
Molecular and Cellular Biology, September 2004, p. 7503-7513, Vol. 24, No. 17
0270-7306/04/$08.00+0 DOI: 10.1128/MCB.24.17.7503-7513.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.
Ludwig Institute for Cancer Research, Stockholm, Sweden
Received 8 March 2004/ Returned for modification 26 April 2004/ Accepted 10 June 2004
 |
ABSTRACT |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
|
INTRODUCTION |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
B/Relish, Cubitus interruptus, and Notch proteins (4, 5, 20). In such instances, nuclear targeting not only is used to provide signals necessary for the correct timing of gene regulation but also provides the means to physically transfer signals from nonnuclear compartments to specific promoter sequences. Fundamental to understanding regulated latent transcription factors is the elucidation of the mechanisms that direct these proteins to change compartments in a controlled fashion. This change may be achieved through a protein modification that regulates the activity of intrinsic nuclear localization determinants; nuclear localization sequences (NLS) are sequence motifs recognized by the nuclear import machinery and, in the opposing manner, nuclear export sequences (NES) are sequence motifs that mediate nuclear export. Although NLS and NES motifs are required for directed nuclear import and export, respectively, perhaps the simplest way to regulate nuclear targeting is through physically tethering or anchoring latent precursor forms of transcription factors outside the nucleus. The first example of such a mechanism is the sterol regulatory element-binding protein (SREBP) (10). SREBP is an integral membrane protein that is anchored in the membranes of the early secretory pathway. The cytoplasmically oriented domain possessing transactivation activity is released from membranes in two successive rounds of proteolytic processing by site-specific membrane-bound proteases (38).
The unicellular budding yeast Saccharomyces cerevisiae was recently found to assess the availability of extracellular nutrients through sensors in the plasma membrane. Sensors, presumably functioning as receptors, that recognize small molecules, such as sugars or amino acids, have been identified (for a review, see reference 18). Ssy1p is a nutrient receptor that functions together with the two peripheral membrane-associated proteins, Ptr3p and Ssy5p, as a sensor of extracellular amino acids. This function was initially demonstrated by the observation that amino acid permease promoters are derepressed upon the addition of small amounts of amino acids (13). Amino acid induction requires all three sensor components, Ssy1p-Ptr3p-Ssy5p (SPS), and the F-box protein encoded by GRR1 (7, 13, 17, 25, 26, 29). The participation of Grr1p in signal transduction is not well understood, but its role as a substrate specificity factor in the Skp1p-Cullin-F-box ubiquitin protein-ligase (SCF) complex suggests a role for ubiquitylation in SPS sensor signal transduction. Consistent with this suggestion, mutants of the SCF complex and ubiquitin have been shown to affect SPS sensor-dependent promoter derepression (7).
Based on several lines of evidence, the homologous zinc finger transcription factors Stp1p and Stp2p are redundant downstream effector components of the SPS sensor pathway. Single deletions of either STP1 or STP2 partially impair SPS sensor signaling, whereas deletions of both fully abolish signaling (1, 12). Stp1p and Stp2p bind to specific upstream activating sequences (UAS) present within SPS sensor-regulated promoters (12, 33). Both Stp1p and Stp2p are synthesized as latent cytoplasmic factors that are activated by receptor-mediated processing (1). In response to the addition of amino acids and in a strictly SPS sensor-dependent manner, Stp1p and Stp2p are endoproteolytically cleaved. This event liberates the DNA-binding and transactivation domains from an approximately 10-kDa N-terminal fragment. The shorter forms of Stp1p and Stp2p accumulate in the nucleus, where they function to transactivate SPS sensor-regulated genes.
In this study, we directly tested the requirement of the proteolytic processing of Stp1p/Stp2p. We found that the N-terminal domains of Stp1p/Stp2p contain two conserved motifs that are required for SPS sensor-dependent regulation. These motifs are functionally distinct and function in parallel to ensure proper nuclear exclusion and proteolytic processing. We also showed that Grr1p is required for the proteolytic processing of Stp1p/Stp2p and that Asi1p, an integral membrane protein, functions to restrict the entry of full-length unprocessed forms of Stp1p and Stp2p into the nucleus. Finally, the N-terminal regulatory domain of Stp1p is transferable and confers full SPS sensor pathway and Asi1p control when fused to an unrelated synthetic transcription factor. Our data clearly indicate that Stp1p and Stp2p are maintained as latent cytoplasmic transcription factors merely because of motifs within their N-terminal regulatory regions.
|
MATERIALS AND METHODS |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Yeast strains.
All yeast strains used in this work (Table 1) are isogenic descendants of the S288c-derived strain AA255/PLY115 (3). Generation of the deletion alleles asi1
8::kanMX, grr150::hphMX4, stp151::Agleu2, stp250::hphMX4, and gap1::PAGP-lacZ has been described elsewhere (1, 16). CAY224 and CAY225 are meiotic segregants obtained from a cross between CAY161 and PLY126. CAY228 was constructed by transforming CAY224 with two PCR products, one containing a 3' portion of kanMX (23) fused to SSY1 flanked by 3' ADE2 genomic sequences and one containing 5' ADE2 genomic sequences fused to PTR3 flanked by a 5' portion of kanMX. Homologous sequences within the region of overlap between the two amplified portions of kanMX facilitated the simultaneous targeting of both fragments to the ADE2 locus (see Fig. 3A). Transformants were selected on G418-supplemented medium, and ade2 (red) colonies carrying PTR3 and SSY1 sequences were identified. Similarly, CAY229 was obtained by transforming CAY225 with two PCR products, one encompassing SSY5 fused to a 5' portion of natMX4 (22) and one containing a 3' portion of natMX4 fused to SHR3. Overhangs in the appropriate primers facilitated targeting to the CAN1 locus (see Fig. 3A). Transformants were selected on clonNAT-supplemented medium and screened for canavanine resistance.
|
|
, ptr3, ssy5, and shr3 alleles. CAY233 is a meiotic segregant, containing both pairs of integration alleles, and was obtained by crossing CAY228 and CAY229. CAY241 is a his3 derivative of CAY233 and was obtained as a meiotic segregant from a cross between PLY861 and CAY233. Plasmids. The plasmids used in this study are listed in Table 2. The mutagenic oligonucleotides and PCR primers used are available on request. Plasmid pCA030 was created by ligating a 7.2-kb HindIII/SalI fragment obtained from YCpAGP1-LacZ (25) with similarly restricted pRS317. The insert of pCA047 was subcloned as an EcoRI/SmaI fragment into pRS317, creating pCA122. Plasmid pCA171 carrying HIS3 was isolated from yeast cells cotransformed with EagI/XhoI-restricted pRS313 and PvuI-restricted pCA047. Plasmid pCA120 was created by single-stranded mutagenesis of pCA047 with oligonucleotide 1 (30). Plasmids pCA127, pCA128, and pCA129 were created by single-stranded mutagenesis of pCA047 with oligonucleotides 2 to 4, respectively. Plasmid pCA135 was created by single-stranded mutagenesis of pCA047 with oligonucleotides 5 and 6. Plasmid pCA161 is a derivative of the commonly used two-hybrid bait plasmid pEG202 (24) with the following two modifications. First, the HIS3 marker was replaced by LYS2 amplified from pRS317 with primers 7 and 8. Second, the first 125 codons of STP1 were introduced in frame (with primers 9 and 10 and pCA029 as a template) (1) with the lexA gene fused to the activation domain (AD) of VP16 (with primers 11 and 12 and pCMXVP16 as a template) (a kind gift from T. Perlmann) (8). Plasmid pCA160 is a derivative of pCA161 created by deleting part of the ADH1 promoter and the entire lexA gene fusion by restriction with SphI and religation. Plasmid pCA212 was created by homologous recombination in yeast cells; a GRR1-containing PCR product (primers 13 and 14) amplified with S288c genomic DNA as a template was cotransformed with KpnI/SacI-restricted pRS316.
|
ß-Galactosidase activity assays. ß-Galactosidase activity was determined with N-lauroyl-sarcosine-permeabilized cells (28). After preincubation of cells in 800 µl of 0.2% (wt/vol) sodium N-lauroyl-sarcosine Z buffer at 30°C for 15 min, a 160-µl aliquot of o-nitrophenyl-ß-D-galactopyranoside (ONPG) solution (4 mg/ml) was added and the tubes were mixed by inversion. The reaction was stopped by the addition of 400 µl of 1 M Na2CO3, and the tubes were centrifuged at 12,000 x g for 5 min. The absorbance of the supernatant was measured at 420 nm. Semiquantitative measurements of ß-galactosidase activity were routinely made by using equally turbid cell suspensions (optical density at 600 nm, 1) diluted 1:1 in 0.4 M potassium phosphate buffer (pH 7) containing 0.2% (wt/vol) sodium N-lauroyl-sarcosine and 0.2 mg of X-Gal/ml. Cell suspensions were incubated at 30°C until blue color was detectable. A similar buffer containing 0.5% (wt/vol) low-melting-point agarose was used as an overlay to assess the ß-galactosidase activity of whole colonies.
Immunoblot analysis. Whole-cell extracts were prepared under denaturing conditions with NaOH and trichloroacetic acid treatment as described previously (35). Extracted proteins were resolved by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) and analyzed by immunoblotting. Immunoblots were incubated with primary antibody diluted in blocking buffer as follows: 12CA5 ascitic fluid (antihemagglutinin [HA] monoclonal antibody), 1:1,000; and anti-LexA polyclonal antibody (Invitrogen), 1:5,000. Immunoreactive bands were visualized by chemiluminescence detection (SuperSignal West Dura extended-duration substrate; Pierce) of horseradish peroxidase conjugated to secondary antibodies (anti-mouse immunoglobulin from sheep and anti-rabbit immunoglobulin from donkey; Amersham) and quantified by using an LAS1000 system (Fuji Photo Film Co. Ltd).
|
RESULTS |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
131 allele (Fig. 1C, REG) (1). Region II corresponds to the predicted site of proteolytic processing (Fig. 1C, scissor). The level of conservation within these regions and their locations suggest that they have important roles in the regulation of Stp1p and Stp2p nuclear exclusion (region I) and endoproteolytic processing (region II).
|
stp2 mutant strain was transformed with plasmids expressing wild-type Stp1p or alanine substitution L65A, F66A, and P67A mutant proteins. Cells were grown in amino-acid-free SD medium, and Stp1p cleavage in whole-cell extracts prepared before and 30 min after induction by leucine was monitored. Leucine induced the proteolytic processing of wild-type Stp1p (Fig. 2A, left panel, compare lanes 1 and 5). Alanine substitution mutations L65A and P67A had negligible effects on processing; the mutant proteins were proteolytically processed as effectively as wild-type Stp1p (Fig. 2A, left panel, compare lanes 2 and 6 and lanes 4 and 8). In contrast, leucine did not induce the proteolytic processing of the F66A mutant protein (Fig. 2A, left panel, compare lanes 3 and 7). The steady-state level of the F66A mutant protein was similar to that of wild-type Stp1p, suggesting that the alanine substitution does not grossly affect protein stability.
|
stp2 mutant strain transformed with a control vector or plasmids expressing wild-type Stp1p or L65A, F66A, and P67A mutant proteins. All strains exhibited robust growth on control SD medium (Fig. 2A, upper right panel). However, on SD medium supplemented with AzC, only cells carrying the control vector or the plasmid expressing the F66A mutant protein were able to grow (Fig. 2A, lower right panel, lanes 1 and 4). The growth of cells expressing the F66A mutant protein demonstrated that this protein is unable to transduce SPS sensor signals. We named the noncleavable and noninducible F66A mutant allele stp1-102 (Fig. 1C). The lack of growth on SD medium supplemented with AzC of cells carrying plasmids expressing wild-type Stp1p or L65A and P67A mutant proteins (Fig. 2A, right panel, lanes 2, 3, and 5) indicates that these proteins are functional, a finding consistent with their amino acid-induced processing.
Alanine mutations in conserved region I constitutively activate Stp1p.
We replaced multiple residues in the branched-chain amino acid cluster with alanine (Fig. 1B, region I). One allele with four alanine substitutions, STP1-133 (Fig. 1C), resulted in phenotypes consistent with constitutive signaling. It suppressed AzC resistance exhibited by an ssy1 strain (Fig. 2B, left panels, compare lanes 2 and 4) and activated the expression of ß-galactosidase from a PAGP1-lacZ reporter plasmid almost to the extent of the constitutive STP1131 allele (Fig. 2B, right panel, compare bar 2 with bars 3 and 4). The steady-state levels of full-length Stp1-133p present in SD medium-grown ssy1 cells were substantially lower than those in the same cells expressing wild-type Stp1p (Fig. 2B, middle panel, lanes 2 and 4), suggesting that the STP1-133 allele may result in misfolding of the N-terminal domain and destabilization of the protein. The fact that the STP1-133 allele phenocopies the characterized STP1131 allele (1) indicates that residues affected by the mutations are important in the retention of unprocessed Stp1p outside the nucleus.
Proteolytic processing is not a prerequisite for transactivation, and full-length Stp1p activates transcription when inappropriately induced to enter the nucleus.
Loss-of-function mutations in the ASI1 gene enable the constitutive expression of Stp1p/Stp2p-dependent genes even in the absence of a functional SPS sensor (16). We previously showed that Stp1p/Stp2p processing does not occur in cells lacking a functional SPS sensor (1); thus, the constitutive expression of Stp1p/Stp2p-dependent genes in asi1 mutants without SPS sensor activity predicts that full-length Stp1p/Stp2p can activate transcription. To examine this possibility, we analyzed the status of Stp1p processing and AzC-resistant growth of asi1 mutant and ASI1 wild-type strains. An stp1 stp2 asi1 strain was transformed with a control plasmid (vector) or plasmids expressing either STP1 or the noncleavable stp1-102 allele. Immunoblot analysis revealed that Stp1p processing was not affected by the loss of ASI1; upon leucine induction, the shorter cleaved form was readily detected (Fig. 2C, left panel, lane 1). In constrast, all of the noncleavable Stp1-102p mutant protein migrated at the position of the full-length unprocessed form (Fig. 2C, left panel, lane 2). The activities of the expressed proteins were examined as AzC resistance. All transformed strains formed colonies of equal sizes on SD medium (Fig. 2C, upper right panel). Cells transformed with the empty plasmid (vector) readily formed colonies on AzC-containing medium (Fig. 2C, lower right panel, lane 1), whereas cells carrying a plasmid expressing wild-type STP1 (Fig. 2C, lower right panel, lane 2) or the stp1-102 allele did not form colonies (Fig. 2C, lower right panel, lane 3). The lack of growth of the STP1-expressing strain is consistent with the observed processing of Stp1p. The finding that stp1 stp2 asi1 cells expressing noncleavable Stp1-102p are unable to grow in the presence of AzC clearly demonstrates that unprocessed Stp1p is able to function as a transactivator if it is able to enter the nucleus.
The observation that the stp1-102 allele encodes a nonprocessed protein that is functional when expressed in an asi1 stp1 stp2 strain but not in an stp1 stp2 strain (Fig. 2C, right panels, lanes 4 and 3, respectively) enabled us to examine the epistasis of mutations in conserved regions I and II (Fig. 1B). We combined the recessive stp1-102 and the dominant STP1-133 mutations into a single gene, creating the double-mutant STP1-102,133 allele. An stp1 stp2 strain was transformed with plasmids expressing noncleavable Stp1-102p, constitutively active Stp1-133p, or double-mutant Stp1-102,133p. Immunoblot analysis showed that Stp1-133p was processed normally but that Stp1-102,133p was not (Fig. 2C, left panel, compare lanes 4 and 5). Cells transformed with the constitutively active STP1-133 allele exhibited AzC sensitivity and failed to form colonies on SD medium supplemented with AzC (Fig. 2C, right panels, lane 5). Also, cells carrying the double-mutant STP1-102,133 allele were unable to grow in the presence of AzC (Fig. 2C, right panels, lane 6). These results demonstrate that in the absence of a functional inhibitory domain (region I), cleavage is not required for unprocessed Stp1p to transactivate gene expression. Thus, it can be concluded that amino acid-induced cleavage is required merely to sever the inhibitory domain from the transactivating portion of Stp1p and that cytoplasmic retention and endoproteolytic activation are functionally separable and distinct processes.
A novel genetic screen identifies recessive mutations in GRR1 that inactivate the SPS sensor pathway. Mutational analysis of the N-terminal region of Stp1p identified the endoproteolytic processing of Stp1p/Stp2p as the key regulatory step in the SPS sensor pathway and prompted us to search for additional factors that may be involved in the processing step. To enable the isolation of rare mutations affecting the SPS sensor pathway, we created a haploid strain harboring functional duplicated alleles of SSY1, PTR3, and SSY5 integrated into the genome (see Materials and Methods) (Fig. 3A). We also chose to duplicate SHR3, a gene encoding a membrane-localized chaperone that is specifically required for the exit of Ssy1p from the endoplasmic reticulum (21, 29). By making our starting strain merodiploid for these genes, our aim was to eliminate the reisolation of recessive loss-of-function mutations in previously identified SPS sensor components. The starting strain also carried an AGP1-promoted lacZ reporter gene integrated into the genome, leading to the expression of ß-galactosidase when the SPS sensor pathway is intact. In contrast, cells with a mutant SPS sensor pathway will lack the expression of detectable ß-galactosidase activity.
Signaling through the SPS sensor pathway leads to the transcription of AGP1 and GNP1, resulting in AzC import and cell cycle arrest (Fig. 1A). Consequently, mutations abolishing SPS sensor signaling make cells AzC resistant. Spontaneously arising AzC-resistant colonies were obtained by plating saturated cultures of the merodiploid starting strain on solid SD medium supplemented with AzC. Six slowly growing AzC-resistant colonies that were small and flat and that remained white after incubation with an X-Gal overlay were identified (Fig. 3B). Under all growth conditions, these six mutants consistently grew at greatly reduced rates compared to the wild-type starting strain. Microscopic analysis revealed that in all instances, the mutant cells were elongate and remained attached to one another, forming branched chains of cells. Consistent with these latter observations, the mutants exhibited a flocculent growth phenotype in liquid cultures.
The growth phenotypes of our newly identified mutant strains closely resemble those exhibited by grr1 mutant strains (14). Complementation analysis was used to address whether the mutations in our strains were indeed allelic with GRR1. All six strains were found to carry recessive mutations; diploids resulting from crosses with the wild-type strain were AzC sensitive and expressed robust ß-galactosidase activity (Fig. 3C). In contrast, diploids from crosses with the grr1 tester strain were AzC resistant and lacked ß-galactosidase activity (Fig. 3C). The lack of complementation of both phenotypes is consistent with the mutations being allelic with GRR1. Genetic linkage was established by sporulating the two diploids obtained from crosses with mutant 1; strict 2:2 segregation was observed in tetrads derived from the cross with the wild-type strain, and no AzC-sensitive spores were recovered from the cross with the grr1 strain. Additionally, plasmid-borne GRR1 complemented the AzC resistance exhibited by mutant 1 (Fig. 3D, compare lanes 1 and 2 with lanes 5 and 6). We conclude that the mutants isolated carry recessive mutations in GRR1.
GRR1 is required for the amino acid-induced endoproteolytic processing of Stp1p.
We previously reported that grr1 mutations do not impair the proteolytic processing of Stp1p, suggesting that Grr1p functions downstream of this regulatory event (1). We investigated whether our newly isolated grr1 mutant alleles affected the proteolytic processing of Stp1p. For wild-type extracts, the full-length form of Stp1p-HA was present in the noninduced control, and the cleaved 10-kDa lower-molecular-mass form was observed after leucine induction (Fig. 4A). In contrast, only the nonprocessed form of Stp1p-HA was found in leucine-induced extracts from the newly isolated grr1 mutants (Fig. 4A).
|
mutant strain. The cleavage of Stp1p-HA in a wild-type strain with an intact SPS sensor and that in an ssy1 mutant strain lacking the receptor component of the SPS sensor were monitored and served as positive and negative controls, respectively. Leucine-induced processing of Stp1p-HA was apparent in the wild-type strain (Fig. 4B, compares lane 1 and 4), whereas no processing was detected in the ssy1 strain (Fig. 4B, compare lanes 2 and 5). Consistent with our analysis of newly isolated grr1 mutant strains, no processing was observed in the grr1 mutant strain (Fig. 4B, compare lanes 3 and 6). Together, these results indicate, contrary to what we previously reported, that GRR1 is required for the proteolytic processing of Stp1p/Stp2p.
Constitutive activation of the Ssy1p receptor does not bypass the GRR1 requirement.
SSY1-102 encodes a dominant mutant form of Ssy1p that constitutively activates the expression of SPS sensor-regulated genes even in the absence of inducing amino acids (19). We tested whether SSY1-102 could suppress the Stp1p-processing defect in grr1 cells. Control wild-type and grr1 strains carrying a plasmid expressing Stp1p-HA were transformed with SSY1- or SSY1-102-containing plasmids and analyzed by immunoblotting. As previously shown, leucine induced Stp1p processing in wild-type cells (Fig. 5A, compare lanes 1 and 5) but not in grr1 cells (Fig. 5A, compare lanes 3 and 7). In uninduced wild-type cells expressing SSY1-102, a portion of immunodetectable Stp1p was found in its processed form (Fig. 5A, lane 2). Interestingly, leucine induction increased the amount of processed Stp1p to a level similar to that observed in SSY1-expressing cells (Fig. 5A, compare lanes 5 and 6). These results account for the constitutive amino acid-independent activation of SPS sensor-regulated genes in SSY1-102-expressing cells. Furthermore, these findings confirm that the proteolytic processing of Stp1p is a direct consequence of the amino acid-induced activation of Ssy1p and is not due to an indirect effect related to amino acid uptake (19). Thus, the constitutive nature of Ssy1-102p signaling appears to be mechanistically similar to amino acid-induced wild-type Ssy1p signaling. The Stp1p-processing defect in the grr1 strain could not be suppressed by the introduction of the SSY1-102 allele even in the presence of leucine (Fig. 5A, compare lanes 4 and 8).
|
133) and the recessive asi1 mutation bypass the requirement for GRR1.
If the lack of Stp1p/Stp2p processing is the underlying reason for the signaling defect in grr1 mutant strains, mutations activating the pathway downstream of the processing step should exert a suppressing effect. To test this possibility, we examined whether the recessive asi1 and dominant activated STP1133 alleles could bypass the need for GRR1. Wild-type, ssy1, and grr1 cells, with and without the asi1 and STP1133 alleles, were spotted on solid SD medium and SD medium supplemented with AzC. All strains grew on the control plate containing SD medium (Fig. 5B, upper panel). In the presence of AzC, the wild-type strain exhibited no growth, whereas the ssy1 and grr1 strains formed colonies, indicating that these strains are defective in SPS sensor signaling (Fig. 5B, lower panel, lanes 2 and 3). The introduction of either asi1 or STP1133 mutations suppressed the loss of SPS sensor activity, as evidenced by the lack of growth of ssy1 asi1 and ssy1 STP1133 strains (Fig. 5B, lower panel, compare lane 2 with lanes 5 and 8). The asi1 and STP1133 mutations also suppressed the grr1 allele (Fig. 5B, lower panel, compare lane 3 with lanes 6 and 9). The epistasis analysis indicates that the defect in SPS sensor signaling exhibited by grr1 cells is identical to that observed in cells carrying loss-of-function mutations in any of SPS sensor component genes (SSY1, PTR3, or SSY5). The N terminus of Stp1p is modular and can be transferred to regulate the activity of an artificial transcription factor. We examined the possibility that the first 125 residues of Stp1p [Stp1(1-125)], including the inhibitory motif (region I) and the cleavage sites (region II), could be transferred to confer amino acid-induced regulation of an artificial transcription factor. The N terminus of Stp1p was fused to a well-characterized synthetic transcription factor comprised of the bacterial LexA DNA-binding protein fused to the strong viral VP16 transcriptional AD. The LexA DNA-binding domain possesses an intrinsic NLS and is constitutively nuclear when expressed in yeast cells (34). A wild-type strain and strains carrying deletions of the SPS sensor components GRR1 and ASI1 were transformed with a plasmid expressing the Stp1(1-125)-LexA-AD fusion protein to assess the full spectrum of SPS sensor pathway regulation.
First, we examined whether the Stp1(1-125)-LexA-AD fusion protein was subject to amino acid-induced SPS sensor-dependent processing. Extracts were prepared from transformed strains and analyzed with anti-LexA antibodies by immunoblotting. In cells grown in SD medium, in the absence of an inducing amino acid, bands corresponding to the full-length Stp1(1-125)-LexA-AD fusion protein were readily detected (Fig. 6A, lane 1). When cells were grown in the presence of leucine, processing was observed in the wild-type and asi1 mutant cells (Fig. 6A, lanes 2 and 12). In these cells, the full-length Stp1(1-125)-LexA-AD fusion protein was detected only weakly, and more intense, faster-migrating bands were observed. No processing was observed in leucine-induced ssy1, ptr3, ssy5, or grr1 mutant cells (Fig. 6A, lanes 3 to 10).
|
, ptr3, ssy5, or grr1 mutant cells (Fig. 6B, lanes 2 to 5). Importantly, the regulation of Stp1(1-125)-LexA-AD derepressed the reporter even in the absence of amino acids in an asi1 strain (Fig. 6B, lane 6). These results indicate that Stp1(1-125) functions as a modular domain that can be transferred to an artificial transcription factor to faithfully confer full amino acid-induced SPS sensor-dependent regulation. Thus, the N-terminal domain is sufficient for both nuclear exclusion and protease recognition. |
DISCUSSION |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
stp2 double mutant, demonstrating that proteolytic removal of the inhibitory domain is strictly required for Stp1p/Stp2p activity. We could exclude the possibility that the lack of transcriptional induction by Stp1-102p was due to a folding defect or a general loss of function, since second site-suppressing mutations placed in cis (STP1-133) or in trans (asi1) enabled the unprocessed mutant form of Stp1p to enter the nucleus and transactivate gene expression (Fig. 2C). The dominant nature and constitutive activity of the STP1-133 allele, which carries mutations within region I, suggest that this motif normally facilitates the cytoplasmic retention of unprocessed Stp1p. The observation that Stp1-133p is proteolytically processed (Fig. 2C), despite the presence of a nonfunctional inhibitory domain, indicates that the N-terminal regulatory domain of Stp1p is modular. These results confirm that the processing of Stp1p/Stp2p is indeed the major regulatory event that transduces SPS sensor-initiated signals, generated at the plasma membrane, to the nucleus and to UASaa-containing promoters. Our analysis revealed that specific and functionally separable motifs within the N-terminal domain provide the molecular basis for the latent nuclear exclusion and amino acid-induced processing of Stp1p/Stp2p. Our current model for Stp1p/Stp2p regulation is presented in Fig. 7. Newly translated Stp1p/Stp2p is excluded from the nucleus by two parallel activities. The primary mechanism is linked to the inhibitory motif designated region I, which appears to anchor the unprocessed full-length forms to an as-yet-undefined cytoplasmic determinant. The secondary mechanism is dependent on the action of Asi1p.
|
The processing defect exhibited by grr1 mutant strains indicates that GRR1 encodes a component that functions in signaling events leading to Stp1p/Stp2p processing. This assignment is supported by our analysis of epistasis relationships. The constitutive activating form of Stp1p, encoded by the dominant STP1131 allele, and mutations in ASI1 were found to efficiently suppress grr1 mutant phenotypes. However, the actual mechanistic role of Grr1p in the SPS sensor signal transduction pathway is difficult to predict based on these newly established genetic relationships. We are currently considering two possibilities; Grr1p either is directly involved in the signal transduction events within the SPS sensor or is a factor required for the activity of one of the SPS sensor components. The latter possibility defines a role for Grr1p without direct involvement in a dynamic signaling process. Based on the previously characterized functions of Grr1p in glucose sensing and cell cycle control (6, 15, 31, 32, 36), it is compelling to imagine a direct role for ubiquitin in the SPS sensor pathway, as previously suggested (7).
The modular nature of the N-terminal region of Stp1p motivated us to examine whether it could be transferred to the well characterized synthetic transactivator LexA-AD. The presence of the N terminus of Stp1p converted this constitutively nucleus-localized factor into a latent fully SPS sensor-regulated factor. This conclusion is based on several observations. First, immunoblot analysis demonstrated that Stp1(1-125)-LexA-AD is endoproteolytically processed in a manner similar to that of full-length Stp1p/Stp2p (Fig. 6A). Furthermore, lexA operator-driven promoter activation was shown to depend on amino acid induction and a functional SPS sensor (Fig. 6B). The OPlexA-lacZ reporter gene was fully repressed in strains carrying mutations in the upstream components of the SPS sensor pathway (ssy1, ptr3, ssy5, and grr1). Finally, constitutive derepression of the OPlexA-lacZ reporter construct was observed in asi1 cells. These data indicate that Stp1(1-125) is able to override the intrinsic NLS within LexA and to confer full SPS sensor pathway control.
The ability of the N-terminal domain of Stp1p to confer the entire known spectrum of SPS sensor pathway control to an unrelated protein supports the notion that all of the regulatory circuits converge on the N-terminal domain. This finding limits the number of feasible mechanisms that may regulate the latency of Stp1p/Stp2p. For example, it is unlikely that the N terminus masks or blocks an intrinsic NLS within Stp1p/Stp2p. Instead, the autonomous function of the N-terminal domain suggests that the inhibitory motif (region I) interacts with a protein that functions as a nuclear exclusion determinant, e.g., a cytoplasmic protein or a component of the nuclear export machinery. If this model proves correct, then the regulatory mechanism exhibits striking similarity with NF-B/Rel signaling; IF-B sequesters NF-B in the cytoplasm and prevents nuclear translocation by binding the actin cytoskeleton via ankyrin repeats (9, 20).
Finally, it has not escaped our attention that the components described here comprise the basic building blocks of a highly specific system for the regulated expression of genes in yeast cells. The exploitation of such a system has some obvious advantages over existing expression systems. Presumably, the activities of other transcription factors (either heterologous, homologous, or completely artificial) can be placed under the control of the SPS sensor by appending the N-terminal domain of Stp1p. Consequently, amino acid-induced gene expression can be restricted to a single synthetic promoter, as we have accomplished with OPlexA-lacZ. Additionally, the transferability of the N-terminal inhibitory domain of Stp1p suggests the possibility of specifically restricting the access of individual proteins to the nucleus in a manner that is readily reversible. This scenario may be useful for testing the physiologic consequences of redirecting and sequestering presumed nuclear factors in the cytoplasm.
|
ACKNOWLEDGMENTS |
|---|
This research was supported by the Ludwig Institute for Cancer Research.
|
FOOTNOTES |
|---|
|
REFERENCES |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
2. Andréasson, C., E. P. A. Neve, and P. O. Ljungdahl. 2004. Four permeases import proline and the toxic proline analogue azetidine-2-carboxylate into yeast. Yeast 21:193-199.[CrossRef][Medline]
3. Antebi, A., and G. R. Fink. 1992. The yeast Ca(2+)-ATPase homologue, PMR1, is required for normal Golgi function and localizes in a novel Golgi-like distribution. Mol. Biol. Cell 3:633-654.[Abstract]
4. Aza-Blanc, P., and T. B. Kornberg. 1999. Ci: a complex transducer of the hedgehog signal. Trends Genet. 15:458-462.[CrossRef][Medline]
5. Baron, M. 2003. An overview of the Notch signalling pathway. Semin. Cell Dev. Biol. 14:113-119.[CrossRef][Medline]
6. Barral, Y., and C. Mann. 1995. G1 cyclin degradation and cell differentiation in Saccharomyces cerevisiae. C. R. Acad. Sci. Ser. III 318:43-50.[Medline]
7. Bernard, F., and B. André. 2001. Ubiquitin and the SCF(Grr1) ubiquitin ligase complex are involved in the signalling pathway activated by external amino acids in Saccharomyces cerevisiae. FEBS Lett. 496:81-85.[CrossRef][Medline]
8. Botling, J., D. S. Castro, F. Oberg, K. Nilsson, and T. Perlmann. 1997. Retinoic acid receptor/retinoid X receptor heterodimers can be activated through both subunits providing a basis for synergistic transactivation and cellular differentiation. J. Biol. Chem. 272:9443-9449.
9. Brivanlou, A. H., and J. E. Darnell, Jr. 2002. Signal transduction and the control of gene expression. Science 295:813-818.
10. Brown, M. S., and J. L. Goldstein. 1997. The SREBP pathway: regulation of cholesterol metabolism by proteolysis of a membrane-bound transcription factor. Cell 89:331-340.[CrossRef][Medline]
11. Burke, D., D. Dawson, and T. Stearns. 2000. Methods in yeast genetics: a Cold Spring Harbor Laboratory course manual. Cold Spring Harbor Laboratory Press, Cold Sping Harbor, N.Y.
12. de Boer, M., P. S. Nielsen, J. P. Bebelman, H. Heerikhuizen, H. A. Andersen, and R. J. Planta. 2000. Stp1p, Stp2p and Abf1p are involved in regulation of expression of the amino acid transporter gene BAP3 of Saccharomyces cerevisiae. Nucleic Acids Res. 28:974-981.
13. Didion, T., B. Regenberg, M. U. Jørgensen, M. C. Kielland-Brandt, and H. A. Andersen. 1998. The permease homologue Ssy1p controls the expression of amino acid and peptide transporter genes in Saccharomyces cerevisiae. Mol. Microbiol. 27:643-650.[CrossRef][Medline]
14. Flick, J. S., and M. Johnston. 1991. GRR1 of Saccharomyces cerevisiae is required for glucose repression and encodes a protein with leucine-rich repeats. Mol. Cell. Biol. 11:5101-5112.
15. Flick, K. M., N. Spielewoy, T. I. Kalashnikova, M. Guaderrama, Q. Zhu, H. C. Chang, and C. Wittenberg. 2003. Grr1-dependent inactivation of Mth1 mediates glucose-induced dissociation of Rgt1 from HXT gene promoters. Mol. Biol. Cell 14:3230-3241.
16. Forsberg, H., M. Hammar, C. Andréasson, A. Moliner, and P. O. Ljungdahl. 2001. Suppressors of ssy1 and ptr3 null mutations define novel amino acid sensor-independent genes in Saccharomyces cerevisiae. Genetics 158:973-988.
17. Forsberg, H., and P. O. Ljungdahl. 2001. Genetic and biochemical analysis of the yeast plasma membrane Ssy1p-Ptr3p-Ssy5p sensor of extracellular amino acids. Mol. Cell. Biol. 21:814-826.
18. Forsberg, H., and P. O. Ljungdahl. 2001. Sensors of extracellular nutrients in Saccharomyces cerevisiae. Curr. Genet. 40:91-109.[CrossRef][Medline]
19. Gaber, R. F., K. Ottow, H. A. Andersen, and M. C. Kielland-Brandt. 2003. Constitutive and hyperresponsive signaling by mutant forms of Saccharomyces cerevisiae amino acid sensor Ssy1. Eukaryot. Cell 2:922-929.
20. Gilmore, T. D. 1999. The Rel/NF-kappaB signal transduction pathway: introduction. Oncogene 18:6842-6844.[CrossRef][Medline]
21. Gilstring, C. F., M. Melin-Larsson, and P. O. Ljungdahl. 1999. Shr3p mediates specific COPII coatomer-cargo interactions required for the packaging of amino acid permeases into endoplasmic reticulum-derived transport vesicles. Mol. Biol. Cell 10:3549-3565.
22. Goldstein, A. L., and J. H. McCusker. 1999. Three new dominant drug resistance cassettes for gene disruption in Saccharomyces cerevisiae. Yeast 15:1541-1553.[CrossRef][Medline]
23. Guldener, U., S. Heck, T. Fielder, J. Beinhauer, and J. H. Hegemann. 1996. A new efficient gene disruption cassette for repeated use in budding yeast. Nucleic Acids Res. 24:2519-2524.
24. Gyuris, J., E. Golemis, H. Chertkov, and R. Brent. 1993. Cdi1, a human G1 and S phase protein phosphatase that associates with Cdk2. Cell 75:791-803.[CrossRef][Medline]
25. Iraqui, I., S. Vissers, F. Bernard, J. O. de Craene, E. Boles, A. Urrestarazu, and B. André. 1999. Amino acid signaling in Saccharomyces cerevisiae: a permease-like sensor of external amino acids and F-box protein Grr1p are required for transcriptional induction of the AGP1 gene, which encodes a broad-specificity amino acid permease. Mol. Cell. Biol. 19:989-1001.
26. Jøorgensen, M. U., M. B. Bruun, T. Didion, and M. C. Kielland-Brandt. 1998. Mutations in five loci affecting GAP1-independent uptake of neutral amino acids in yeast. Yeast 14:103-114.[CrossRef][Medline]
27. Kellis, M., N. Patterson, M. Endrizzi, B. Birren, and E. S. Lander. 2003. Sequencing and comparison of yeast species to identify genes and regulatory elements. Nature 423:241-254.[CrossRef][Medline]
28. Kippert, F. 1995. A rapid permeabilization procedure for accurate quantitative determination of beta-galactosidase activity in yeast cells. FEMS Microbiol. Lett. 128:201-206.[Medline]
29. Klasson, H., G. R. Fink, and P. O. Ljungdahl. 1999. Ssy1p and Ptr3p are plasma membrane components of a yeast system that senses extracellular amino acids. Mol. Cell. Biol. 19:5405-5416.
30. Kunkel, T. A., J. D. Roberts, and R. A. Zakour. 1987. Rapid and efficient site-specific mutagenesis without phenotypic selection. Methods Enzymol. 154:367-382.[Medline]
31. Li, F. N., and M. Johnston. 1997. Grr1 of Saccharomyces cerevisiae is connected to the ubiquitin proteolysis machinery through Skp1: coupling glucose sensing to gene expression and the cell cycle. EMBO J. 16:5629-5638.[CrossRef][Medline]
32. Mosley, A. L., J. Lakshmanan, B. K. Aryal, and S. Özcan. 2003. Glucose-mediated phosphorylation converts the transcription factor Rgt1 from a repressor to an activator. J. Biol. Chem. 278:10322-10327.
33. Nielsen, P. S., B. van den Hazel, T. Didion, M. de Boer, M. Jørgensen, R. J. Planta, M. C. Kielland-Brandt, and H. A. Andersen. 2001. Transcriptional regulation of the Saccharomyces cerevisiae amino acid permease gene BAP2. Mol. Gen. Genet. 264:613-622.[CrossRef][Medline]
34. Rhee, Y., F. Gurel, Y. Gafni, C. Dingwall, and V. Citovsky. 2000. A genetic system for detection of protein nuclear import and export. Nat. Biotechnol. 18:433-437.[CrossRef][Medline]
35. Silve, S., C. Volland, C. Garnier, R. Jund, M. R. Chevallier, and R. Haguenauer-Tsapis. 1991. Membrane insertion of uracil permease, a polytopic yeast plasma membrane protein. Mol. Cell. Biol. 11:1114-1124.
36. Skowyra, D., K. L. Craig, M. Tyers, S. J. Elledge, and J. W. Harper. 1997. F-box proteins are receptors that recruit phosphorylated substrates to the SCF ubiquitin-ligase complex. Cell 91:209-219.[CrossRef][Medline]
37. Thompson, J. D., D. G. Higgins, and T. J. Gibson. 1994. CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res. 22:4673-4680.
38. Wang, X., R. Sato, M. S. Brown, X. Hua, and J. L. Goldstein. 1994. SREBP-1, a membrane-bound transcription factor released by sterol-regulated proteolysis. Cell 77:53-62.[CrossRef][Medline]
This article has been cited by other articles:
| |||||||||
A (pCA127), F66
