Multiple inputs control sulfur-containing amino acid synthesis in Saccharomyces cerevisiae

Abstract

In Saccharomyces cerevisiae, transcription of the MET regulon, which encodes the proteins involved in the synthesis of the sulfur-containing amino acids methionine and cysteine, is repressed by the presence of either methionine or cysteine in the environment. This repression is accomplished by ubiquitination of the transcription factor Met4, which is carried out by the SCF(Met30) E3 ubiquitin ligase. Mutants defective in MET regulon repression reveal that loss of Cho2, which is required for the methylation of phosphatidylethanolamine to produce phosphatidylcholine, leads to induction of the MET regulon. This induction is due to reduced cysteine synthesis caused by the Cho2 defects, uncovering an important link between phospholipid synthesis and cysteine synthesis. Antimorphic mutants in S-adenosyl-methionine (SAM) synthetase genes also induce the MET regulon. This effect is due, at least in part, to SAM deficiency controlling the MET regulon independently of SAM's contribution to cysteine synthesis. Finally, the Met30 protein is found in two distinct forms whose relative abundance is controlled by the availability of sulfur-containing amino acids. This modification could be involved in the nutritional control of SCF(Met30) activity toward Met4.

FIGURE 1:

Metabolic pathways. (A) Synthesis of sulfur-containing amino acids in S. cerevisiae. The conversion of SAM to SAH occurs through myriad cellular methylation reactions, of which only three are shown in B. (B) Synthesis of phosphatidylcholine in S. cerevisiae. In addition, ethanolamine, monomethylethanolamine, dimethylethanolamine, and choline can be converted to phosphatidylethanolamine, phosphatidylmonomethylethanolamine (phosphatidyl-MME), phosphatidyldimethylethanolamine (phosphatidyl-DME), and phosphatidylcholine, respectively, through the action of the Kennedy pathway (not shown).

FIGURE 2:

CHO2 mutations induce MET3-GFP. All histograms are of GFP fluorescence from cells carrying MET3-GFP under its native promoter, on a log₁₀ scale, as determined by flow cytometry. For all histograms, WT refers to a methionine prototrophic (MET15) strain with MET3-GFP (JRY9355), grown overnight with or without methionine as indicated. (A) MET3-GFP fluorescence in met15∆ cells carrying a nonsense mutation in CHO2 (JRY9359; left) or a complete deletion of CHO2 (JRY9360; right), grown in media containing 134 μM (20 μg/ml) methionine. (B) Fluorescence imaging of a tetrad dissection of a cho2∆/CHO2 met15∆/MET15 MET3-GFP/MET3-GFP diploid grown on plates with 1 mM methionine. The darkness of a colony corresponds to its intensity of GFP fluorescence. Colonies with a heavy circle are met15∆, whereas colonies with a dotted circle are MET15. (C) Fluorescence imaging of a tetrad dissection of cho2∆ met15∆ and CHO2 met15∆ colonies with MET3-GFP, replica plated to a plate with 1 mM methionine and no glutathione (top) or a plate with 1 mM methionine + 1 mM glutathione (bottom). (D) Fluorescence imaging of a tetrad dissection of cho2∆ met15∆ MET3-GFP (JRY9360) crossed to met6∆ from the MATα Yeast Knockout Collection grown on plates with 1 mM methionine. (E) MET3-GFP fluorescence in cho2∆ cells with nutritional remediation by compounds that form phospholipids downstream of Cho2’s action. MME, monomethylethanolamine; DME, dimethylethanolamine. (F) MET3-GFP fluorescence in cho2∆ opi3∆ cells (JRY9361) with nutritional remediation by compounds that form phospholipids downstream of Cho2’s action. (The cho2∆ opi3∆ cells were fed choline in addition to the pathway intermediates, as they required choline for growth.)

FIGURE 3:

SAM synthetase mutations induced MET3-GFP. (A) Fluorescence imaging of tetrad dissections of four mutants (JRY9391, JRY9394, JRY9395, and JRY9396) backcrossed to an unmutagenized strain (JRY9355 or JRY9356) and grown on a plate with 1 mM methionine. A total of 10–15 tetrads were analyzed, and the colonies selected for the pooling are marked. (B) Flow cytometry of sam1∆ MET3-GFP cells carrying either the wild-type SAM1 allele (JRY9469) or the SAM1-G310D allele (JRY9470) on a single-copy plasmid expressed from the native SAM1 promoter and grown with 134 μM methionine. (C) Flow cytometry of sam1∆ or sam2∆ cells with MET3-GFP (JRY9508 and JRY9509, respectively) grown in 134 μM methionine as compared with SAM1-G310D cells. (D) Flow cytometry of SAM1-G310D cells with MET3-GFP grown with 134 μM methionine and carrying either the wild-type SAM1 allele on a single-copy plasmid expressed from the native SAM1 promoter (JRY9454) or a blank single-copy plasmid with no insert (JRY9472). (E) Flow cytometry of SAM1-G310D cells with MET3-GFP grown in 134 μM methionine, as well as with additional 0.2 mM SAM or 10 mM glutathione. (F) Fluorescence imaging of a tetrad dissection of SAM2-C93Y/SAM2 met6Δ/MET6 MET3/MET3-GFP diploids grown on a plate with 1 mM methionine. Ten tetrads were analyzed.

FIGURE 4:

The Met30 protein had two isoforms. (A) Immunoblot of Met30-3xHA expressed from a single-copy plasmid in met15Δ cells (JRY9354) grown with 134 μM methionine or after growth for 1 h in medium containing neither methionine nor cysteine, prepared either as a whole-cell extract or by direct immunoprecipitation of HA. Detection of Pgk1 was used as a loading control, and an isogenic strain lacking HA-tagged Met30 was used as a negative control. (B) RNA structure, as predicted using mfold (Zuker, 2003), in a section of the MET30 mRNA (bases 178–214). (C) Conservation of a predicted RNA structure in MET30 of other species in the sensu stricto clade. DNA sequence is shown, with bracket notation for the S. cerevisiae RNA shown above. Aligned DNA sequence for the other species of the sensu stricto clade are shown below (Scannell et al., 2011; Liti et al., 2013), with mutations highlighted. (Saccharomyces uvarum is also referred to as S. bayanus var. uvarum or S. bayanus; Libkind et al., 2011.) Green highlighting indicates that the opposite arm of the stem loop contains a compensating mutation that preserves the predicted hairpin structure. Yellow highlighting indicates that the mutation was not compensated but still allows pairing with its predicted partner. Red highlighting indicates a mutation to a base unable to pair with the opposite arm. Note that because S. uvarum anchors the tree, it is not possible to determine whether differences in its sequence arose along its lineage or along the lineage shared by the other members. Those differences are notated as occurring along the S. uvarum lineage for simplicity. A similar caveat applies to the mutations at −11 and +11. (D) Mutational scheme to disrupt potential hairpin formation. The translation of the targeted segment is shown above the sequence. (E) Immunoblot of met15Δ cells carrying either wild-type Met30-3xHA (JRY9354) or Met30-3xHA with hairpin-disrupting mutations (JRY9510) expressed from single-copy plasmids and grown either in medium with 134 μM methionine or after growth for 1 h in medium lacking both methionine and cysteine. (F) MET3 expression determined by RT-PCR in MET15 cells with wild-type or hairpin-disrupted MET30 alleles (JRY9511 and JRY9512, respectively) expressed off single-copy plasmids and grown overnight with or without 134 μM methionine. All cDNA values were internally normalized to ACT1 cDNA values from the same cDNA preparation and normalized to wild type grown with methionine afterward. n = 2; error bars show SE.

FIGURE 5:

Posttranslational modification profiling of Met30. (A) Diagram of the Met30 amino acid sequence with known domains and residues conserved to C. albicans highlighted. C. albicans sequence was obtained from the Saccharomyces Genome Database (http://yeastgenome.org), and alignment was done with ClustalW2 (Goujon et al., 2007). Residues detected as modified are circled. (B) MET3 expression determined by RT-PCR in met30∆ MET15 cells carrying single-copy plasmids with wild-type or mutant MET30 alleles grown overnight with 134 μM methionine or in medium lacking both methionine and cysteine. WT refers to JRY9490, C95A to JRY9491, C95D to JRY9492, C95Y to JRY9505, C455A to JRY9494, C455D to JRY9495, C95,455A to JRY9497, and C95,455D to JRY9498. MET3 cDNA values were internally normalized to ACT1 cDNA values from the same cDNA preparation and then normalized to wild type grown with methionine. n = 2 for wild type, C95,455A, and C95,455D; n = 1 for the others. Error bars show SE. (C) Immunoblot of wild-type or mutant Met30-3xHA proteins expressed from single-copy plasmids in met15∆ cells either grown with 134 μM methionine or after growth for 1 h in medium without methionine or cysteine. Wild type refers to JRY9477, C95,455A to JRY9484, and C95,455D to JRY9485. n = 2; the increased intensity of Met30-3xHA in C94,455A did not replicate. (D) Dissections of met30∆/MET30 met15∆/MET15 ura3∆/ura3∆ diploids (JRY9451) carrying single-copy URA3 plasmids carrying mutant alleles of MET30.