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Suppressors of ssy1 and ptr3 Null Mutations Define Novel Amino Acid Sensor-Independent Genes in Saccharomyces cerevisiae
Hanna Forsberg1,a, Mårten Hammar1,2,a, Claes Andréassona, Annalena Molinér3,a, and Per O. Ljungdahlaa Ludwig Institute for Cancer Research, S-171 77 Stockholm, Sweden
Corresponding author: Per O. Ljungdahl, Ludwig Institute for Cancer Research, Nobels väg 3, Box 240, S-171 77 Stockholm, Sweden., plju{at}licr.ki.se (E-mail)
Communicating editor: A. P. MITCHELL
 | ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Ssy1p and Ptr3p are components of the yeast plasma membrane SPS amino acid sensor. In response to extracellular amino acids this sensor initiates metabolic signals that ultimately regulate the functional expression of several amino acid-metabolizing enzymes and amino acid permeases (AAPs). As a result of diminished leucine uptake capabilities, ssy1
leu2 and ptr3 leu2 mutant strains are unable to grow on synthetic complete medium (SC). Genes affecting the functional expression of AAPs were identified by selecting spontaneous suppressing mutations in amino acid sensor-independent (ASI) genes that restore growth on SC. The suppressors define 11 recessive (asi) complementation groups and 5 dominant (ASI) linkage groups. Strains with mutations in genes assigned to these 16 groups fall into two phenotypic classes. Mutations in the class I genes (ASI1, ASI2, ASI3, TUP1, SSN6, ASI13) derepress the transcription of AAP genes. ASI1, ASI2, and ASI3 encode novel membrane proteins, and Asi1p and Asi3p are homologous proteins that have conserved ubiquitin ligase-like RING domains at their extreme C termini. Several of the class II genes (DOA4, UBA1, BRO1, BUL1, RSP5, VPS20, VPS36) encode proteins implicated in controlling aspects of post-Golgi endosomal-vacuolar protein sorting. The results from genetic and phenotypic analysis indicate that SPS sensor-initiated signals function positively to facilitate amino acid uptake and that two independent ubiquitin-mediated processes negatively modulate amino acid uptake.
THE ability of Saccharomyces cerevisiae cells to rapidly respond and adapt to changing environmental conditions is essential for viability. A prerequisite for generating a proper physiological response is the ability to sense and subsequently transduce information regarding the extra- and intracellular environments. Sensor-initiated signals are used to make dynamic adjustments in patterns of gene expression and protein turnover, processes that enable cells to express the necessary components appropriate for prevailing conditions. Recently, several plasma membrane (PM) nutritional sensors were identified in yeast that monitor nutrient availability in the extracellular environment. These include Snf3p and Rgt2p, two glucose sensors (
Ssy1p is a member of the amino acid permease (AAP) family that has a unique 200-amino-acid N-terminal extension that is required for sensor activity (
It was demonstrated that SPS sensor-mediated signals are required for amino acid-induced transcription of several AAP genes (i.e., AGP1, BAP3, GNP1, BAP2, VAP1, and TAT2), the peptide transporter gene PTR2, and the arginase gene CAR1 (
Several genes required for transcription of SPS-controlled AAP genes were identified; they include STP1, STP2, ABF1, UGA35/DAL81, GRR1, TUP1, and LEU3 (
Ubiquitin-mediated processes are also known to regulate the activity of many AAPs at the post-translational level (
In this report, we present a genetic selection designed to isolate mutations that restore amino acid uptake in SPS sensor-deficient strains. Due to impaired uptake of leucine, strains carrying ssy1 and ptr3 null mutations in combination with a leu2 auxotrophic allele are unable to grow in media containing complex mixtures of amino acids (
| MATERIALS AND METHODS |
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| TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Media and strains:
Standard media, including YPAD, sporulation, ammonia-based synthetic minimal dextrose (SD) supplemented as required to enable growth of auxotrophic strains, and synthetic complete dextrose (SC), were prepared as described (
The strains used in this work (Table 1) are descendants of isogenic strains PLY115 and PLY118 (identical to strains AA255 and AA288, respectively; 13 are meiotic segregants from a cross between PLY115 and HKY20. Strains YMH151 and YMH158 carrying ptr315 are meiotic segregants from a cross between PLY115 and HKY31. To obtain ASI1-, ASI3-, and BRO1-deleted strains (YMH349, CAY10, and YMH351, respectively), PCR-produced asi18::loxP-kanMX-loxP, asi31::loxP-kanMX-loxP, and bro165::loxP-kanMX-loxP deletion constructs were used to transform the haploid strain YMH119. Null mutant strains were selected on the basis of their exhibiting both G418 resistance and SC-positive growth phenotypes. A PCR-produced asi29::hisG-URA3-hisG deletion construct was used to transform the haploid strain YMH119 to uracil prototrophy and SC-positive growth, resulting in strain YMH343. To create the bul150::hisG-URA3-kanr-hisG deletion strain CAY2, YMH119 was transformed with pCA007 digested with NotI and SalI. Double recombinants were selected on SD with required auxotrophic supplements lacking uracil. Sensitivity to 20 µM cadmium chloride in SC was used to verify bul1 phenotype of deletion strains (
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Cloning of ASI1, ASI2, and ASI6:
The synthetic slow-growth phenotype on SD medium displayed by asi1 asi6, asi2 asi6, and asi3 asi6 double mutant strains was used to clone ASI1, ASI2, and ASI6 (see Fig 5). Strains YMH367 (asi1-1 asi6-1) and YMH373 (asi2-1 asi6-2) were transformed with a genomic plasmid library (
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ASI1:
Three plasmids that complemented the SC phenotype of strain YMH213 but not that of strain YMH245 were isolated. The DNA sequences at the ends of the inserts in these plasmids were different but from the same chromosomal region. A trimmed plasmid carrying only the open reading frame (ORF) YMR119w (pMH423) complemented asi1 mutant phenotypes. An asi18::kanMX deletion strain exhibited phenotypes identical to those of other asi1 strains, and a diploid strain resulting from the crossing of YMH349 (ssy113 leu2-3,112 asi18:: kanMX) and YMH214 (ssy113 leu2-3,112 asi1-1) grew on SC. All spores derived from this diploid were SC+, and G418 resistance segregated 2:2.
ASI2:
Four plasmids complemented the SC phenotype when transformed into YMH217 but not when transformed into YMH257. These plasmids contained overlapping sequences containing the ORF YNL159c. Plasmid pMH51 carrying only YNL159c complemented the asi2 mutant phenotypes. A strain (YMH343) carrying a precise deletion of YNL159c was constructed and found to share the same phenotypes as those of asi2 mutants. The diploid resulting from crossing YMH343 (ssy113 leu2-3,112 asi29::hisG-URA3-hisG) and YMH218 (ssy113 leu2-3,112 asi2-1) grew well on SC. All spores derived from this diploid were SC+, and Ura+ segregated 2:2.
ASI6:
The inserts of plasmids complementing only the asi6 mutations in YMH257 or YMH245 were sequenced and found to contain sequences including the BRO1 gene (pMH66). These plasmids complemented phenotypes associated with asi6 mutations upon retransformation. A bro165::kanMX deletion strain was constructed (YMH351). This strain, in which the entire open reading frame of BRO1 is removed, exhibited asi6 mutant phenotypes. A diploid obtained from crossing YMH351 (ssy113 leu2-3,112 bro165::kanMX) and YMH246 (ssy113 leu2-3,112 asi6-1) grew well on SC. The spores resulting from this diploid were all SC+, and G418 resistance segregated 2:2.
Cloning of ASI3 and the identification of BUL1 as a multicopy suppressor of asi3 mutations:
Despite repeated attempts using numerous plasmid libraries to clone ASI3 by complementing the slow-growth phenotype of an asi3 asi6-2 double mutant strain (YMH376) on SD (Fig 7), we failed to identify uniquely complementing plasmids. As was the case during the cloning of ASI1 and ASI2, plasmids containing LEU2 and UBI4 were isolated. We therefore attempted to clone ASI3 directly using an alternative phenotype. In contrast to the ssy1 leu2 strain (YMH119), the ssy1 leu2 asi3 strain (YMH237) is sensitive to AzCOOH (Fig 4A, compare dilution series 2 with 6) and able to grow on SC (Fig 1, compare dilution series 2 with 6). Strain YMH237 was transformed with a CEN-based plasmid library (
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Due to difficulties of isolating a bona fide asi3 complementing plasmid we suspected that expression of ASI3, or sequences in the vicinity of the ASI3 locus, is toxic when introduced into Escherichia coli. Therefore, we constructed a library from genomic yeast DNA and transformed it directly into yeast. DNA from strain S288C was prepared as described (
Selection for clones resistant to AzCOOH yielded two plasmids, pCA011 and pCA012, containing overlapping genomic fragments containing the ORF YNL008c. When passaged in E. coli these plasmids displayed toxicity, and plasmid-containing bacterial colonies were extremely slow growing and appeared translucent. Upon reintroduction into YMH237 both plasmids simultaneously conferred AzCOOH resistance and inhibited growth on SC. An asi31::kanMX deletion allele that removes the entire coding sequence of YNL008c was created and used to construct strain CAY10. A diploid strain obtained from crossing CAY10 (ssy113 leu2-3,112 asi31::kanMX) and YMH238 (ssy113 leu2-3,112 asi3-1) grew well on SC and was sensitive to AzCOOH, and spore-derived colonies resulting from this diploid were all SC+; G418 resistance segregated 2:2.
Cloning of ASI4, ASI5, ASI7, ASI8, ASI9, ASI10, and ASI11:
Strains YMH241 (asi4-1), YMH209 (asi5-1), YMH291 (asi7-1), YMH253 (asi8-1), YMH221 (asi9-1), YMH229 (asi9-2), YMH303 (asi10-1), and YMH293 (asi11-1) were transformed with a genomic plasmid library (
Plasmid construction and DNA manipulations:
Plasmids and oligonucleotides used in this study are listed in Table 2. Plasmid inserts were sequenced using primers specific for the T3 and T7 promoter sequences or YCp50 flanking sequences. Plasmids containing ASI1-containing sequences are diagrammed in Fig 2. Plasmid pMH4 was made by inserting the 2.7-kb BsrBI fragment from an asi1 complementing plasmid containing YMR119w, including 426-bp upstream and 395-bp downstream sequences of YMR119w (including YMR119w-a), into pRS316 (PinAI, pMH4BamHI, pMH4HindIII, and pMH4SacI, respectively. Plasmid pMH423 contains the minimal 2.3-kb HpaI/SacI-complementing fragment of ASI1 cloned into SmaI/SacI-digested pRS426 (
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The asi18::kanMX, asi29::URA3, and bro165::kanMX deletion cassettes were amplified by PCR. Primer pairs prASID-1 and prASI1D-2, prASI3D-1 and prASI3D-2, and prBRO1D-1 and prBRO1D-2 were used to generate fragments encompassing a loxP-kanMX-loxP cassette flanked on both sides by upstream and downstream regions of ASI1, ASI3, and BRO1, respectively. Plasmid pUG6 (9::URA3 fragment encompassing the URA3 gene flanked on both sides by hisG and DNA upstream and downstream (of the first and last codon, respectively) of YNL159c using the plasmid pMPY-ZAP as template (
Northern blotting analysis:
Strains YMH121, YMH119, YMH151, YMH213, YMH217, YMH237, YMH241, YMH209, YMH245, YMH291, YMH253, YMH221, YMH331, YMH333, YMH249, YMH233, YMH336, YMH337, and YMH335 were transformed with pRS315 (
-32P]dCTP (3000 Ci/mmol; Amersham, Little Chalfont, UK) using the random-primed DNA labeling kit (MBI Fermentas Molecular Biology) and purified using Bio Spin columns (Bio-Rad Laboratories, Hercules, CA). After hybridization, blots were rinsed once with 5x SSC, 0.1% SDS (1x SSC is 0.15 M NaCl, 0.015 M sodium citrate), washed two times with 5x SSC, 0.1% SDS, and one time with 0.1x SSC, 0.1% SDS. Washes were performed at 55° for 20 min. After washings, blots were visualized and quantified using a Fujix Bio-Image analyzer BAS1500 (Fuji Photo Film Co., Tokyo).
| RESULTS |
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| TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Isolation of mutations that suppress ssy1 and ptr3 null alleles:
As a result of diminished leucine uptake capabilities, ssy1 and ptr3 mutant strains carrying the auxotrophic leu2-3,112 allele are unable to grow on SC. The synthetic lethality on SC is likely to be due to the high amino acid content of this medium; the overabundance of competing amino acids interfere with the residual uptake mechanisms, effectively inhibiting uptake of the required amino acid. Thus, when grown on SC, leu2 auxotrophic ssy1 or ptr3 mutants cannot synthesize leucine nor can they import leucine from the external environment at rates sufficient to support growth. Spontaneous mutations capable of suppressing the synthetic growth defect of ssy113 leu2-3,112 and ptr315leu2-3,112 strains on SC media (Fig 1, dilution series 2 and 3) were sought in parallel. Six different colonies from ssy113 leu2-3,112 strains YMH119 (MATa) and YMH126 (MAT) were inoculated in 12 separate tubes containing SD medium, and the cultures were grown to OD600 of 4. Aliquots (100 µl) of each culture were individually spread on separate SC plates, and SC+ colonies were picked after 3 days. The same procedure was used to isolate suppressor mutants from cultures of ptr315 leu2-3,112 strains YMH151 (MATa) and YMH158 (MAT).
To determine whether the suppressing mutations were dominant or recessive, each suppressor mutant was backcrossed to its respective wild-type strain of opposite mating type, YMH119 or YMH126 for ssy1 suppressors and YMH151 or YMH158 for ptr3 suppressors. The diploid strains were sporulated, and tetrad analysis was used to examine whether the observed suppression resulted from mutations in single genes. In each case the suppressing phenotype segregated 2:2. Backcrossed spore-derived suppressor strains of both mating types were saved and used for subsequent phenotypic characterization. In total, 23 recessive and 8 dominant mutations were isolated in the ssy1 background, and 19 recessive and 9 dominant mutations were isolated in the ptr3 background (Table 3).
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Complementation tests were initially performed by mating the MATa ssy1 or ptr3 suppressed strains with the corresponding ssy1 or ptr3 MAT suppressed strains. Diploid strains were selected on SD media and after replica plating to SC complementation was assessed. Within each complementation group, only one mutant strain of those isolated from the same starting culture was saved. In the cases where suppressing mutations were isolated in only one mating type, backcrossed spore-derived suppressor strains were used to complete the complementation analysis. Crosses between dominant suppressor mutants were subjected to tetrad analysis, which enabled us to determine whether the mutations were allelic.
ssy1 and ptr3 suppressing mutations define 16 ASI genes:
We investigated if the ssy1-suppressing mutations were able to suppress the SC growth phenotype exhibited by ptr3 leu2 strains. The analysis required two separate crosses. First, a member from each complementation group (ssy1 sup leu2) was crossed to an SSY1 PTR3 leu2 strain. After sporulation, nonparental ditype tetrads were identified, and the two SC+ spore-derived colonies were assumed to result from SSY1 PTR3 sup haploids. The sup allele was then moved into a ptr3 genetic background by crosses to an appropriate ptr314::URA3 leu2 strain (either YMH153 or YMH166). After tetrad dissection on permissive medium, spore-derived colonies were assessed for their ability to grow on SC. In each case Ura+ SC+ spores were recovered at frequencies expected for two independently segregating genes. These results indicate that although the suppressing mutations were isolated in a ssy1 leu2 genetic background they suppressed the synthetic lethality exhibited by ptr314::URA3 leu2 strains equally well. In an analogous manner we examined whether the ptr3 suppressing mutations were capable of suppressing the SC growth phenotype of ssy1 leu2 strains. As was the case for the ssy1 suppressing mutations, each of the ptr3 suppressors suppressed ssy1 mutations equally well.
These results allowed us to investigate whether the ssy1 and ptr3 suppressors were mutations in the same genes. Complementation analysis indicated that the combined set of mutants define 11 recessive complementation groups and 5 dominant linkage groups. On the basis of the overlap of suppression we designated the affected genes ASI (Table 3). Dilution series of wild-type, ssy1, and ptr3 deleted cells, as well as cells carrying a ssy1 deletion in combination with suppressor mutations, were spotted onto SD and SC media. All strains grew equally well on SD medium (Fig 1). On SC, wild-type cells grow, whereas ssy1 and ptr3 null mutant cells do not (Fig 1, compare dilution series 1 with 2 and 3). ssy1 null mutant strains carrying mutant alleles of each of the ASI genes grew well on SC (Fig 1, dilution series 4–20).
Recessive asi mutations identify three novel and eight previously characterized genes:
ASI genes complementing the recessive asi mutations were isolated (see MATERIALS AND METHODS). Table 3 summarizes the identity of the cloned genes. ASI1 is a novel gene that has not previously been described. The ASI1 locus within complementing plasmid pMH4 contains two overlapping open reading frames (Fig 2). The 212 most 3' bases of ORF YMR119w overlap with ORF YMR119w-a. To unambiguously determine which ORF corresponds to ASI1, we introduced frameshift mutations at four different positions, generating plasmids pMH4PinAI, pMH4BamHI, pMH4HindIII, and pMH4SacI (Fig 2). Strain YMH349 (ssy113 leu2-3,112 asi18:: kanMX), lacking both YMR119w and YMR119w-a, and strain YMH213 (ssy113 leu2-3,112 asi1-1) were transformed with pMH4 and the frame-shifted plasmid derivatives. Only plasmids pMH4 and pMHSacI complemented the asi18::kanMX deletion and asi1-1 mutations. The minimal HpaI-SacI genomic fragment in pMH423, which was found to complement asi1 mutations, ends 52 bases downstream of the YMR119w stop codon and removes the 111 bp of the 3'-end of YMR119w-a. These results indicate that ASI1 is identical to ORF YMR119w (Fig 2). ASI2 and ASI3 correspond to previously uncharacterized ORFs YNL159c and YNL008c, respectively.
Eight of the recessive asi mutations were complemented by previously described genes (Table 3). Isolation of asi mutations residing in these known genes indicates that mutations affecting transcriptional regulation and ubiquitination/protein degradation, as well as vesicular transport, are able to suppress ssy1 and ptr3 deficiencies. ASI4 and ASI5 were complemented by TUP1 and SSN6, respectively. Tup1p and Ssn6p are two physically interacting components of a general repressor complex of RNA polymerase II transcription (
ASI1, ASI2, and ASI3 encode novel polytopic membrane proteins:
ASI1 and ASI3 encode homologous proteins that share a high degree of structural similarity. Asi1p and Asi3p are 24% identical and 39% similar over the entire length of their amino acid sequence, 624 and 669 amino acid residues, respectively. Structural predictions based on the aligned sequences suggest that Asi1p and Asi3p are integral polytopic membrane proteins with five membrane-spanning domains (Fig 3A;
ASI2 encodes a novel protein of 289 amino acids with a calculated molecular weight of 33 kD. The protein contains three putative membrane-spanning regions (Fig 3B;
Growth characteristics of amino acid sensor-independent mutant strains:
Strains carrying null alleles of SSY1 and PTR3 are known to be sensitive to high concentrations of lysine and resistant to the toxic proline analog AzCOOH (
In comparison to wild-type cells, ssy1 and ptr3 null mutant cells are AzCOOH resistant (Fig 4A, compare dilution series 1 with 2 and 3). ssy1 strains carrying suppressing asi1, asi2, asi3, tup1, ssn6, and ASI13-1 mutations exhibit wild-type sensitivity to AzCOOH (Fig 4A, dilution series 4–8 and 17). Strains carrying suppressing mutations in DOA4, UBA1, RSP5, ASI12, and ASI14 appear to grow less well than the nonsuppressed single ssy1 strain (Fig 4A, compare dilution series 2 with series 10–13, 16, and 18). In contrast, ssy1 strains carrying suppressing mutations in BRO1, VPS20, VPS36, ASI15, and ASI16 exhibit enhanced resistance to AzCOOH (Fig 4A, compare dilution series 1 with series 9, 14, 15, 19, and 20).
Suppressing mutations exert differential transcriptional effects:
The ability of asi mutations to restore transcriptional defects associated with ssy1 null mutations was investigated in strains grown under suppressing conditions in SC medium (Fig 4B). To enable all strains to grow under these conditions, wild-type, ssy1, and ptr3 strains, and individual ssy1 strains carrying suppressor mutations were transformed with a plasmid carrying the LEU2 gene (pRS315). Cells were cultured in liquid SC (without leucine) to an OD600 of 0.8 and harvested. Total RNA was prepared and expression of GNP1, BAP2, AGP1, and GAP1 was analyzed by Northern blotting. In wild-type cells the expression of GNP1 and BAP2 was readily detected, the expression of AGP1 was also observed but comparatively weak, and GAP1 expression was below the level of detection (Fig 4B, lane 2). In ssy1 and ptr3 mutant cells the pattern of expression appeared reversed. In these cells GAP1 expression was readily detected, whereas GNP1, BAP2, and AGP1 expression was not (Fig 4B, lanes 1 and 3). In comparison to ssy1 strains, ssy1 strains carrying asi1, asi2, asi3, tup1, ssn6, and ASI13-1 mutations had elevated levels of both GNP1 and BAP2 transcripts (Fig 4B, compare lane 1 with lanes 4–8 and 16). GNP1 transcription was also slightly increased in ssy1 bro1 and ssy1 doa4 mutants (Fig 4B, lanes 9 and 10), and BAP2 expression was readily observed in ssy1 vps20 and ssy1 vps36 strains (Fig 4B, lanes 13 and 14, respectively). Mutations in ASI1, ASI2, ASI3, and ASI13 derepressed the transcription of AGP1 (Fig 4B, lanes 4–6 and 16); AGP1 expression in these strains was higher than in wild-type cells (Fig 4B, lane 2). In wild-type cells grown in ammonium-based SC media the transcription of GAP1 was repressed (Fig 4B, lane 2). As previously described (
bro1 mutations show synthetic interactions with asi1, asi2, asi3, rsp5, and ASI16-1 mutations:
Synthetic interactions between several amino acid sensor-independent mutations were uncovered during complementation analysis (Table 4). ssy1 null mutant strains carrying asi1-1, asi2-1, asi3-1, ASI16-1, rsp5-91, bro1-61, and bro1-62 mutations grow well on SC and SD (Fig 5, dilution series 1–7). However, ssy1 bro1-62 strains carrying either asi1-1, asi2-1, asi3-1, or ASI16-1 mutations exhibited impaired growth on SD (Fig 5, dilution series 8–11). The same phenomenon was observed when these suppressor mutations were introduced into a ssy1 bro1-61 background. The impaired growth observed on SD constituted the actual basis for cloning ASI1, ASI2, and BRO1 (see MATERIALS AND METHODS). Interestingly, in ssy1 strains rsp5 mutations showed allele-specific interactions with bro1 mutations; rsp5-91 exhibited the slow-growth phenotype on SD when combined with bro1-61 (Fig 5, dilution series 12) and bro1-62, whereas rsp5-92 combined with either bro1-61 or bro1-62 did not (Table 4).
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Genetic interactions were also observed between tup1 and ssn6 mutations (Table 4). Diploids generated by crossing YMH242 (ssy1 tup1-41) and YMH209 (ssy1 ssn6-51) grew well on SC medium. The growth of these strains on SC is an example of unlinked noncomplementation (
Expression of BUL1 decreases amino acid uptake in a dose-dependent manner:
BUL1-containing plasmids, originating from two different genomic libraries, were repeatedly isolated during attempts to clone the ASI3 gene (see MATERIALS AND METHODS). These plasmids complemented the AzCOOH-sensitive phenotype exhibited by a ssy1 asi3 strain (Fig 4A, dilution series 6). To ascertain if the observed complementation was specific for asi3 mutations we transformed wild-type (PLY118), asi1-1 (YMH213), asi2-1 (YMH217), asi3-1 (YMH237), and ASI13-1 (YMH233) strains with low-copy (BUL1) or multicopy (2µ-BUL1) plasmids. Dilution series of transformants were spotted onto minimal SD medium (without uracil) containing 100 µg/ml of AzCOOH. Wild-type as well as each of the respective ssy1 asi mutant strains transformed with a vector control were sensitive to AzCOOH (Fig 6A, dilution series 1, 4, 7, 10, and 13). BUL1 expressed from either the low-copy or multicopy plasmid conferred AzCOOH resistance in all strains (Fig 6A). When expressed in the wild-type strain (PLY118), BUL1 exhibited a clear gene dosage effect (Fig 6A, compare dilution series 2 and 3).
BUL1 was similarly expressed in a wild-type (PLY118) and an ssy1 null mutant strain (YMH119). Dilution series of transformants were spotted onto SD medium supplemented with required amino acids (Fig 6B). Expression of BUL1 from a low-copy or a high-copy plasmid did not affect growth of wild-type cells (Fig 6B, dilution series 1–3). However, the expression of BUL1 in the ssy1 null mutant strain inhibited growth (Fig 6B, compare dilution series 4 with 5 and 6). This result suggests that the residual amino acid uptake systems that remain operative in the absence of SSY1 are inhibited by BUL1 overexpression.
A bul1 null allele exhibits a dominant amino acid sensor-independent phenotype:
To determine whether a bul1 mutation functions as an amino acid sensor-independent suppressor, a bul1 null mutant strain was constructed in a ssy1 leu2 background. The ability of ssy1 leu2 (YMH119) and ssy1 leu2 bul1 (CAY2) haploid strains, as well as ssy1/ssy1 leu2/leu2 (YMH119 x YMH126), ssy1/ssy1 leu2/leu2 BUL1/bul1 (YMH126 x CAY2) diploid strains, to grow on SD and SC media was analyzed. All strains grew well on SD medium (Fig 6C, dilution series 1, 2, 5, and 6). Neither the ssy1 haploid nor homozygous ssy1/ssy1 diploid strains grew on SC (Fig 6C, dilution series 3 and 7). In contrast, the haploid ssy1 bul1 double mutant strain exhibited robust growth on SC (Fig 6C, series 4), indicating that the bul1 null mutation gives rise to an amino acid sensor-independent phenotype. Dilutions of the BUL1/bul1 heterozygous diploid strain resulting from the cross YMH126 x CAY2 showed intermediate growth on SC (Fig 6C, series 8). The dominant nature of the bul1 null mutation was independently checked by picking several colonies of the BUL1/bul1 heterozygous diploid growing nonselectively on SD and streaking the cells on SC. In each of the resulting streaks, colonies formed that were uniform in size, confirming that bul1 null mutation acts in a dominant manner to suppress ssy1 null mutations. It was previously reported that bul1 null mutations exhibit dominant phenotypes with respect to isoflurane resistance (
ASI12 is allelic to BUL1:
Dominant mutations in ASI12 were recurrently isolated (Table 3), suggesting that the ASI12-suppressing alleles are the consequence of loss-of-function mutations. The finding that a null allele of BUL1 suppressed ssy1-related phenotypes in a dominant manner raised the possibility that ASI12 is allelic to BUL1. We tested this possibility in two ways. First, we integrated URA3 immediately 5' of BUL1 by transforming strain YMH119 with NarI-digested pAH001. The resulting strain, AHY1 (ssy1 leu2 BUL1::URA3), carries a functional BUL1 and is unable to grow on SC. AHY1 was crossed to YMH250 (ssy1 leu2 ASI12-1), the resulting diploid was sporulated, and tetrads were dissected on SD (supplemented with the necessary auxotrophic requirements). The ability to grow on SC and the URA3 marker segregated 2:2 in an opposing manner; i.e., no SC+ Ura+ recombinant spores were recovered, indicating that ASI12-1 and BUL1 are genetically linked. Second, we crossed SC+ strains CAY2 (ssy1 leu2 bul150::URA3) and YMH250 (ssy1 leu2 ASI12-1). The resulting diploid was sporulated, and tetrads were dissected. All spore-derived colonies were able to grow on SC, confirming that ASI12 is allelic to BUL1.
| DISCUSSION |
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| TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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We isolated 59 independent spontaneously arising mutations that enable ssy1 leu2 and ptr3 leu2 strains to grow on SC media. These mutations defined 16 ASI genes. The phenotypes of strains carrying mutations in ASI genes fall into two classes. Mutations in the class I genes (ASI1, ASI2, ASI3, TUP1, SSN6, ASI13) derepress the transcription of AAP genes. In contrast the class II suppressor mutations exhibit minimal or no transcriptional effects. Several of the cloned class II genes (DOA4, RSP5, VPS20, VPS36) encode proteins that participate in controlling aspects of the post-Golgi endosomal-vacuolar protein sorting pathway. On the basis of the number and distribution of mutant alleles (Table 3), we expect that the screen is not saturated and assume that mutations in other genes could be identified that suppress the leucine uptake defects of ssy1 leu2 and ptr3 leu2 strains.
ASI1, ASI2, and ASI3 encode membrane-associated components of a ubiquitin-based regulatory system that negatively modulates the transcription of AAP genes:
Recessive mutations in ASI1, ASI2, and ASI3 derepress the transcription of AAP genes (Fig 4B). The recessive nature of these mutations suggests that the wild-type gene products, Asi1p, Asi2p, and Asi3p, normally function as negative modulators of AAP gene transcription. Mutations in these genes enable ssy1 leu2 and ptr3 leu2 strains to grow on SC (Fig 1). This growth phenotype indicates that the increased AAP gene transcription results in increased capacity to take up amino acids. Consistent with this conclusion, these mutant strains regain sensitivity to the toxic amino acid analog AzCOOH (Fig 4A).
Strains carrying the dominant ASI13-1 mutation exhibit similar growth phenotypes (Fig 4A) and pattern of AAP transcription (Fig 4B) as observed in asi1, asi2, and asi3 mutant strains. The ASI13-1 mutation manifests a more enhanced level of suppression, i.e., more robust growth on SC and higher levels of AAP gene transcription. The finding that mutations in these four class I ASI genes exhibit similar phenotypes raises the possibility that the respective gene products function within a single pathway. The dominant nature of the ASI13-1 mutation indicates that the altered gene product acts independently of both SPS sensor-mediated regulation and Asi1p/Asi2p/Asi3p-dependent function. The dominant suppressing activity of the ASI13-1 allele is independent of ASI1, ASI2, and ASI3 since an asi1/asi1 ASI13-1/ASI13 diploid displays the more intense ASI13-1 mutant phenotypes (M. HAMMAR, unpublished observations). This latter finding suggests that Asi13p acts downstream of Asi1p, Asi2p, and Asi3p.
What is the function of ASI1 and ASI3? The presence of a RING domain in the C terminus of Asi1p and Asi3p suggests that these proteins belong to a superfamily of E3 ubiquitin ligases (for recent reviews see
The predicted structural features of Asi1p and Asi3p, the five membrane-spanning domains and the C-terminal localized RING domain, are strikingly similar to those found in Hrd1p/Der3p (
The class II ASI gene products Bul1p, Rsp5p, and Bro1p are components of a ubiquitin-based regulatory system that downregulates AAPs:
We found that BUL1 functions as a dose-dependent suppressor of amino acid uptake, and multiple copies of BUL1 pleiotropically complement asi1, asi2, asi3, and ASI13-1 mutations (Fig 6A). The pleiotropic nature of the observed complementation suggests that Bul1p functions downstream of, or in a parallel pathway with, Asi1p, Asi2p, Asi3p, and Asi13p. Significantly, the original BUL1 (ASI12) alleles isolated in the selection for amino acid sensor-independent genes and a BUL1 null allele act in a dominant manner to facilitate amino acid uptake (Fig 6C). The dose dependency of BUL1 expression suggests that Bul1p is a limiting factor that normally functions to negatively regulate amino acid uptake.
Rsp5p and Bul1p are known to physically interact as part of large protein complex (
In addition to its role in downregulating PM proteins, Rsp5p is known to participate in the ubiquitination of numerous proteins involved in a wide variety of diverse cellular processes.