Lysine Starvation Research Articles

Synthetic lethality between toxic amino acids, RTG-target genes and chaperones in Saccharomyces cerevisiae.

The toxicity of non-proteinogenic amino acids has been known for decades. Numerous reports describe their antimicrobial/anticancer potential. However, these molecules are often toxic to the host as well; thus, a synthetic lethality approach that reduces the dose of these toxins while maintaining toxicity can be beneficial. Here we investigate synthetic lethality between toxic amino acids, the retrograde pathway, and molecular chaperones. In Saccharomyces cerevisiae, mitochondrial retrograde (RTG) pathway activation induces transcription of RTG-target genes to replenish alpha-ketoglutarate and its downstream product glutamate; both metabolites are required for arginine and lysine biosynthesis. We previously reported that tolerance of canavanine, a toxic arginine derivative, requires an intact RTG pathway, and low-dose canavanine exposure reduces the expression of RTG-target genes. Here we show that only a few of the examined chaperone mutants are sensitive to sublethal doses of canavanine. To predict synthetic lethality potential between RTG-target genes and chaperones, we measured the expression of RTG-target genes in canavanine-sensitive and canavanine-tolerant chaperone mutants. Most RTG-target genes were induced in all chaperone mutants starved for arginine; the same trend was not observed under lysine starvation. Canavanine exposure under arginine starvation attenuated and even reversed RTG-target-gene expression in the tested chaperone mutants. Importantly, under nearly all tested genetic and pharmacological conditions, the expression of IDH1 and/or IDH2 was induced. In agreement, idh1 and idh2 mutants are sensitive to canavanine and thialysine and show synthetic growth inhibition with chaperone mutants. Overall, we show that inhibiting molecular chaperones, RTG-target genes, or both can sensitize cells to low doses of toxic amino acids.

Lysosomal membrane transporter purification and reconstitution for functional studies.

Lysosomes achieve their function through numerous transporters that import or export nutrients across their membrane. However, technical challenges in membrane protein overexpression, purification, and reconstitution hinder our understanding of lysosome transporter function. Here, we developed a platform to overexpress and purify the putative lysine transporter Ypq1 using a constitutive overexpression system in protease- and ubiquitination-deficient yeast vacuoles. Using this method, we purified and reconstituted Ypq1 into proteoliposomes and showed lysine transport function, supporting its role as a basic amino acid transporter on the vacuole membrane. We also found that the absence of lysine destabilizes purified Ypq1 and causes it to aggregate, consistent with its propensity to be downregulated in vivo upon lysine starvation. Our approach may be useful for the biochemical characterization of many transporters and membrane proteins to understand organellar transport and regulation.

Chaperoning transmembrane helices in the lipid bilayer.

Elimination of membrane proteins often requires recognition of their transmembrane domains (TMDs) in the lipid bilayer. In this issue, Arines et al. (2020. J. Cell Biol.https://doi.org/10.1083/jcb.202001116) show that in Saccharomyces cerevisiae, the vacuole-associated Rsp5 ubiquitin ligase uses a TMD in substrate adaptor Ssh4 to recognize membrane helices in Ypq1, which targets this lysine transporter for lysosomal degradation during lysine starvation.

A selective transmembrane recognition mechanism by a membrane-anchored ubiquitin ligase adaptor

While it is well-known that E3 ubiquitin ligases can selectively ubiquitinate membrane proteins in response to specific environmental cues, the underlying mechanisms for the selectivity are poorly understood. In particular, the role of transmembrane regions, if any, in target recognition remains an open question. Here, we describe how Ssh4, a yeast E3 ligase adaptor, recognizes the PQ-loop lysine transporter Ypq1 only after lysine starvation. We show evidence of an interaction between two transmembrane helices of Ypq1 (TM5 and TM7) and the single transmembrane helix of Ssh4. This interaction is regulated by the conserved PQ motif. Strikingly, recent structural studies of the PQ-loop family have suggested that TM5 and TM7 undergo major conformational changes during substrate transport, implying that transport-associated conformational changes may determine the selectivity. These findings thus provide critical information concerning the regulatory mechanism through which transmembrane domains can be specifically recognized in response to changing environmental conditions.

Reconfiguration of Transcriptional Control of Lysine Biosynthesis in Candidaalbicans Involves a Central Role for the Gcn4 Transcriptional Activator.

Evolution of transcriptional control is essential for organisms to cope with diversification into a spectrum of environments, including environments with limited nutrients. Lysine biosynthesis in fungi occurs in eight enzymatic steps. In Saccharomycescerevisiae, amino acid starvation elicits the induction of LYS gene expression, mediated by the master regulator Gcn4 and the pathway-specific transcriptional regulator Lys14. Here, we have shown that the activation of LYS gene expression in the human fungal pathogen Candidaalbicans is predominantly controlled by Gcn4 under amino acid starvation conditions. Multiple lines of study showed that the four C.albicans LYS14-like genes have no role in the regulation of lysine biosynthesis. Whereas Gcn4 is dispensable for the growth of S.cerevisiae under lysine deprivation conditions, it is an essential regulator required for the growth of C.albicans under these conditions, as gcn4 deletion caused lysine auxotrophy. Gcn4 is required for the induction of increased LYS2 and LYS9 mRNA but not for the induction of increased LYS4 mRNA. Under lysine or isoleucine-valine deprivation conditions, Gcn4 recruitment to LYS2 and LYS9 promoters was induced in C.albicans. Indeed, in contrast to the S.cerevisiae LYS gene promoters, all LYS gene promoters in C.albicans harbored a Gcn4 binding site but not all harbored the S.cerevisiae Lys14 binding site, indicating the evolutionary divergence of cis-regulatory motifs. Thus, the transcriptional rewiring of the lysine biosynthetic pathway in C.albicans involves not only neofunctionalization of the four LYS14-like genes but the attendant strengthening of control by Gcn4, indicating a coordinated response with a much broader scope for control of amino acid biosynthesis in this human pathogen. IMPORTANCE Microbes evolve rapidly so as to reconfigure their gene expression to adapt to the metabolic demands in diverse environmental niches. Here, we explored how conditions of nutrient deprivation regulate lysine biosynthesis in the human fungal pathogen Candidaalbicans. We show that although both Saccharomyces cerevisiae and C.albicans respond to lysine deprivation by transcriptional upregulation of lysine biosynthesis, the regulatory factors required for this control have been reconfigured in these species. We found that Gcn4 is an essential and direct transcriptional regulator of the expression of lysine biosynthetic genes under lysine starvation conditions in C.albicans. Our results therefore suggest that the regulation of the lysine biosynthetic pathway in Candida clade genomes involves gain of function by the master transcriptional regulator Gcn4, coincident with the neofunctionalization of the S.cerevisiae pathway-specific regulator Lys14.

Ammonium-Dependent Shortening of CLS in Yeast Cells Starved for Essential Amino Acids Is Determined by the Specific Amino Acid Deprived, through Different Signaling Pathways

Ammonium (NH4 +) leads to chronological life span (CLS) shortening in Saccharomyces cerevisiae BY4742 cells, particularly evident in cells starved for auxotrophy-complementing amino acids (leucine, lysine, and histidine) simultaneously. Here, we report that the effect of NH4 + on aging yeast depends on the specific amino acid they are deprived of. Compared with no amino acid starvation, starvation for leucine alone or in combination with histidine resulted in the most pronounced NH4 +-induced CLS shortening, whereas starvation for lysine, alone or in combination with histidine resulted in the least sensitivity to NH4 +. We also show that NH4 +-induced CLS shortening is mainly mediated by Tor1p in cells starved for leucine or histidine but by Ras2p in cells starved for lysine, and in nonstarved cells. Sch9p protected cells from the effect of NH4 + under all conditions tested (starved or nonstarved cells), which was associated with Sch9p-dependent Hog1p phosphorylation. Our data show that NH4 + toxicity can be modulated through manipulation of the specific essential amino acid supplied to cells and of the conserved Ras2p, Tor1p, and Sch9p regulators, thus providing new clues to the development of environmental interventions for CLS extension and to the identification of new therapeutic targets for diseases associated with hyperammonemia.

Molecular Mechanisms Underlying the Positive Stringent Response of the Bacillus subtilis ilv-leu Operon, Involved in the Biosynthesis of Branched-Chain Amino Acids

Branched-chain amino acids are the most abundant amino acids in proteins. The Bacillus subtilis ilv-leu operon is involved in the biosynthesis of branched-chain amino acids. This operon exhibits a RelA-dependent positive stringent response to amino acid starvation. We investigated this positive stringent response upon lysine starvation as well as decoyinine treatment. Deletion analysis involving various lacZ fusions revealed two molecular mechanisms underlying the positive stringent response of ilv-leu, i.e., CodY-dependent and -independent mechanisms. The former is most likely triggered by the decrease in the in vivo concentration of GTP upon lysine starvation, GTP being a corepressor of the CodY protein. So, the GTP decrease derepressed ilv-leu expression through detachment of the CodY protein from its cis elements upstream of the ilv-leu promoter. By means of base substitution and in vitro transcription analyses, the latter (CodY-independent) mechanism was found to comprise the modulation of the transcription initiation frequency, which likely depends on fluctuation of the in vivo RNA polymerase substrate concentrations after stringent treatment, and to involve at least the base species of adenine at the 5' end of the ilv-leu transcript. As discussed, this mechanism is presumably distinct from that for B. subtilis rrn operons, which involves changes in the in vivo concentration of the initiating GTP.

Global gene expression profiling of wild type andlysCknockoutEscherichia coliW3110

Aspartokinase III, encoded by lysC, is responsible for the first step of lysine biosynthesis in Escherichia coli. In this study, a lysC knockout E. coli W3110 strain was generated to study the differential gene expression profiles of wild type and lysC knockout strains. Several significant changes were observed, including biosynthesis of lysine, oxaloacetate, alpha-ketoglutarate and glutamate genes. Genes related to transporters and heat shock proteins were also affected by lysC knockout. The results indicated that the lysC knockout strain exhibited some phenomena similar to lysine starvation. The data generated by this study further clarify the systematic role of lysC in lysine biosynthesis.

Leader Peptide-mediated Transcriptional Attenuation of Lysine Biosynthetic Gene Cluster in Thermus thermophilus

The molecular mechanism for regulation of the genes involved in the biosynthesis of amino acids is poorly identified in Thermus thermophilus. In this study, we analyzed the transcriptional control of the major lysine biosynthetic gene cluster in T. thermophilus. S1 nuclease mapping revealed that the transcription, which is repressed by lysine, starts at 111 bp, upstream of the translational start codon, ATG, for the homocitrate synthase (hcs) gene. The 5'-leader region of 111 bp carries a sequence that can encode a short peptide of 14 amino acids with tandem-arranged lysine residues in its sequence. The nucleotide sequence of the region suggests that the transcript can form complicated secondary structures. Deletion of most of the 5'-leader region or mutation of the tandem lysine codons suppressed the transcriptional repression by lysine. Mutation of the tandem codons from lysine to glutamine resulted in glutamine-dependent repression of the gene connected downstream, indicating that the leader peptide mediated the transcriptional attenuation of the gene expression. This is the first report demonstrating the transcriptional regulation of amino acid biosynthesis in T. thermophilus.

Starvation for an essential amino acid induces apoptosis and oxidative stress in yeast

Protracted starvation of auxotrophic Saccharomyces cerevisiae strains for an essential amino acid is commonly used to allow investigation of adaptive mutation mechanisms during starvation-induced cell cycle arrest. Under these conditions, the majority of cells dies during the first 6 days. We investigated starving cells for markers of programmed cell death and for the production of reactive oxygen species (ROS). We observed that protracted starvation for lysine or histidine resulted in an increasing number of cells exhibiting DNA fragmentation and chromatin condensation, thus an apoptotic phenotype. Not only respiration-competent cells but also respiratory deficient rho 0 cells were able to undergo programmed cell death. In addition the starving cells rapidly exhibited indicators of oxidative stress, independently of their respiratory competence. These results indicate that starvation for an essential amino acid results in severe cell stress, which may finally be the trigger of programmed cell death.

Novel chimeric spermidine synthase-saccharopine dehydrogenase gene (SPE3-LYS9) in the human pathogen Cryptococcus neoformans.

The Cryptococcus neoformans LYS9 gene (encoding saccharopine dehydrogenase) was cloned and found to be part of an evolutionarily conserved chimera with SPE3 (encoding spermidine synthase). spe3-lys9, spe3-LYS9, and SPE3-lys9 mutants were constructed, and these were auxotrophic for lysine and spermidine, spermidine, and lysine, respectively. Thus, SPE3-LYS9 encodes functional spermidine synthase and saccharopine dehydrogenase gene products. In contrast to Saccharomyces cerevisiae spe3 mutants, the polyamine auxotrophy of C. neoformans spe3-LYS9 mutants was not satisfied by spermine. In vitro phenotypes of spe3-LYS9 mutants included reduced capsule and melanin production and growth rate, while SPE3-lys9 mutants grew slowly at 30 degrees C, were temperature sensitive in rich medium, and died upon lysine starvation. Consistent with the importance of saccharopine dehydrogenase and spermidine synthase in vitro, spe3-lys9 mutants were avirulent and unable to survive in vivo and both functions individually contributed to virulence. SPE3-LYS9 mRNA levels showed little evidence of being influenced by exogenous spermidine or lysine or starvation for spermidine or lysine; thus, any regulation is likely to be posttranscriptional. Expression in S. cerevisiae of the full-length C. neoformans SPE3-LYS9 cDNA complemented a lys9 mutant but not a spe3 mutant. However, expression in S. cerevisiae of a truncated gene product, consisting of only C. neoformans SPE3, complemented a spe3 mutant, suggesting possible modes of regulation. Therefore, we identified and describe a novel chimeric SPE3-LYS9 gene, which may link spermidine and lysine biosynthesis in C. neoformans.

Loss of Compartmentalization Causes Misregulation of Lysine Biosynthesis in Peroxisome-Deficient Yeast Cells

To characterize the metabolic role of peroxisomes in yeast cells under physiological conditions, we performed a comprehensive meta-analysis of published microarray data. Previous studies of yeast peroxisomes have mainly been focused on the function of peroxisomes under extreme conditions, such as growth on oleate or methanol as the sole carbon source, and may therefore not be representative of the normal physiological role of yeast peroxisomes. Surprisingly, our analysis of the microarray data reveals that the only pathway responding to peroxisome deficiency in mid-log phase is lysine biosynthesis, whereas classical peroxisomal pathways such as beta-oxidation are unaffected. We show that the upregulation of lysine biosynthesis genes in peroxisome-deficient yeasts shares many characteristics with the physiological response to lysine starvation. We provide data that suggest that this is the result of a "pathological" stimulation of the Lys14p transcriptional activator by the pathway intermediate aminoadipate semialdehyde. Mistargeting of the peroxisomal lysine pathway to the cytosol increases the active concentration of aminoadipate semialdehyde, which is no longer contained in the peroxisome and can now activate Lys14p at much lower levels than in wild-type yeasts. This is the first well-documented example of pathway misregulation in response to peroxisome deficiency and will be useful in understanding the phenotypic details of human peroxisome-deficient patients (Zellweger syndrome).

A single point mutation of hamster aminoacyl-tRNA synthetase causes apoptosis by deprivation of cognate amino acid residue.

We have isolated a series of temperature-sensitive mutants for cell-proliferation from the BHK21 cell line derived from the golden hamster (Nishimoto & Basilico 1978; Nishimoto et al. 1982). Using these mutants as a recipient of DNA-mediated gene transfer, we have been cloning human genes which complement these ts mutants. Cultures of tsBN269 cells, a temperature-sensitive mutant of the BHK21 cell line, underwent apoptosis at 39.5 degrees C, a nonpermissive temperature. The gene complementing the tsBN269 cells was cloned and found to encode lysyl-tRNA synthetase. Indeed, tsBN269 cells were found to have a single cytosine to a thymine point mutation at the first nucleotide of codon 542 in hamster lysyl-tRNA synthetases. Due to this mutation, the activity of lysyl-tRNA synthetase was reduced--even at 33.5 degrees C, a permissive temperature. Consistent with these findings, while supplementation with lysine permitted tsBN269 cells to grow at a nonpermissive temperature, the deprivation of lysine caused apoptosis in tsBN269 cells, even at 33.5 degrees C. Cycloheximide inhibited the apoptosis caused by lysine starvation at 33.5 degrees C, but not at 39.5 degrees C. We also found that another hamster temperature-sensitive mutant, tsBN250, which is defective in histidyl-tRNA synthetase, entered apoptosis with the deprivation of histidine. Our data suggested that the defect in aminoacyl-tRNA synthetase turned on the cascade of apoptosis that was already present in the cells.

Relationship between guanosine tetraphosphate and accuracy of translation in Salmonella typhimurium

In bacteria a high level of mistranslation is observed in amino acid starved rel-, but not rel+, strains, and mistranslation can be studied qualitatively by means of "stuttering" experiments in two-dimensional protein gels. It has been suggested that the low level of mistranslation that occurs in rel+ strains is assured by guanosine 5'-diphosphate 3'-diphosphate (ppGpp), a nucleotide whose intracellular concentration greatly increases in rel+ cells under amino acid starvation. In the present study the relationship between level of ppGpp and mistranslation was analyzed by performing stuttering experiments in amino acid starved bacteria that contained either high or low levels of ppGpp. Three strains of Salmonella typhimurium were used in these experiments: a relA+ hisT+ strain (TA997), a relA+ hisT strain (TA1001), and a relA hisT strain (PD2). These strains were first characterized with respect to macromolecular syntheses and ppGpp levels under exponential growth and under amino acid starvation. Both rel+ strains exhibited stringent control over RNA synthesis. ppGpp accumulated to high levels when TA997 was starved for either of three amino acids. Starvation of TA1001 for histidine did not cause accumulation of ppGpp, whereas starvation for lysine and arginine produced high levels of ppGpp. Extracts from the three strains, obtained either under exponential growth or under amino acid starvation, were then subjected to two-dimensional electrophoretic anaylsis: mistranslation was observed whenever ppGpp was absent. In particular, starvation of TA1001 for histidine resulted in high mistranslation frequencies, while under lysine and arginine starvation mistranslation was undetectable, regardless of whether the cells were rel+ or rel-.(ABSTRACT TRUNCATED AT 250 WORDS)

Control points in Neurospora crassa nuclear division cycle: different effects of the inhibition of protein accumulation.

The correlation between protein synthesis and the nuclear division cycle in Neurospora crassa hyphae was studied by inhibiting protein accumulation by two different experimental procedures: (1) starvation for lysine in a lysine-requiring mutant (lys-1); and (2) addition of cycloheximide. Lysine starvation in a lys-1 strain of N. crassa quickly blocked the nuclear division cycle and nuclei accumulated in G1 phase, as judged by their DNA content. After re-addition of lysine to starved cultures, a discontinuous pattern of uridine incorporation into DNA can be seen, showing that the nuclei were well synchronized. On the other hand, treatment with cycloheximide caused the arrest of a large proportion of the nuclei, also, in the G2 phase of the cell cycle. These results indicate that inhibition of protein synthesis may have multiple effects on the cell cycle in N. crassa and that, while moderate inhibition specifically blocks nuclei at a regulatory point in late G1, strong or complete inhibition demonstrates requirement for protein synthesis at other points in the cycle that are not necessarily regulatory points.

Allgemeine Kontrolle der Aminosäurebiosynthese in Mutanten von Candida spec. EH 15/D

The general control of amino acid biosynthesis was investigated in Candida spec. EH 15/D, using single and double mutant auxotrophic strains and prototrophic revertants starved for their required amino acids. These experiments show that starvation for lysine, histidine, arginine, leucine, threonine, proline, serine, methionine, homoserine, asparagine, glutamic acid or aspartic acid can result in derepression of enzymes. A correlation was found between the degree of derepression, growth of strains, and concentration of required amino acids. The amino acids pcol pattern of mutants and revertants is different from that in the wild type strain.

Cross-pathway control of ornithine carbamoyltransferase synthesis in Neurospora crassa.

The pattern of cross-pathway regulation of the arginine synthetic enzyme ornithine carbamoyltransferase was investigated in Neurospora crassa, using single and double mutant auxotrophic strains starved for their required amino acids. These experiments show that starvation for histidine, tryptophan, isoleucine, valine or arginine can result in derepression of ornithine carbamoyltransferase. Methionine starvation also gave slight derepression, but starvation for lysine or leucine gave little or no effect.

Investigation of the role of uncharged tRNA in the regulation of polypeptide chain initiation by amino acid starvation in cultured mammalian cells; a reappraisal.

The rate of initiation of protein synthesis in mammalian cells in culture is regulated by the availability of essential amino acids. Previous reports have suggested that this effect is mediated by changes in the ratio of uncharged to charged tRNA species in these cells. We have investigated this possibility by examining the formation of 40‐S subunit. Met‐tRNAf ribosomal initiation complexes in extracts of Ehrlich ascites tumour cells; this is the stage in initiation previously shown to be regulated in response to lysine starvation of the intact cells. Exposure of cells to a low concentration of cycloheximide makes polypeptide chain elongation the ratelimiting step, allows reformation of polysomes in starved cells and abolishes the effect of lysine deprivation on overall protein synthesis. Treatment with a high concentration of cycloheximide inhibits all protein synthesis and ‘freezes’ the polysomes in the state of aggregation which exists in the cell at that moment. The latter conditions also permit the tRNA pool to become fully charged in both fed and lysine‐starved cells. In cell‐free extracts prepared from cells treated with either low or high concentrations of cyclolieximide 40‐S initiation complex formation nevertheless remains defective after lysine starvation of the cells. Addition of deacylated tRNA, oxidized with periodate to prevent its recharging, to initiating cell‐free extracts inhibits neither 40‐S initiation complex formation nor overall protein synthesis in vitro. Addition of lysine to an extract from lysine‐starved cells, under conditions where this amino acid is charged on to endogenous tRNA, fails to stimulate either 40‐S initiation complex formation or protein synthesis in vitro. These results suggest that amino acid starvation regulates the formation of 40‐S polypeptide chain initiation complexes in mammalian cells by a mechanism which is not influenced by the rate of protein synthesis and does not directly involve uncharged tRNA.

Regulation of lysine- and lysine-plus-threonine-inhibitable aspartokinases in Bacillus brevis.

Further studies on the expression of the two aspartokinase activities in Bacillus bovis are presented. Aspartokinase I (previously shown to be inhibited and repressed by lysine) was found to be repressed by diaminopimelate in the wild-type strain. However, in a mutant unable to convert diaminopimelate to lysine, starvation for lysine resulted in an increase in aspartokinase I activity. Thus, lysine itself or an immediate metabolite was the true effector of repression. Aspartokinase II (previously shown to be inhibited by lysine plus threonine) was repressed by threonine. Studies with the parent strain and auxotrophs inidicated that only threonine or an immediate metabolite of threonine was involved in this repression. Methionine and isoleucine were not effectors of any of the detected aspartokinase activities. Apart from inhibition and repression controls, a third as yet undefined regulatory mechanism operated to decrease the levels of both aspartokinases as growth declined, even in mutants in which repression control was absent. In thiosine-resistant, lysine-excreting mutants with elevated levels of aspartokinase, the increase in activity could always be attributed to one enzyme or the other, never both. The existence of separate structural genes for each aspartokinase is therefore suggested.

"Two out of three" codon reading leading to mistranslation in vivo.

Strains of Escherichia coli were starved for asparagine or lysine in order to increase the in vivo level of mistranslation. In a relA strain, asparagine starvation increased the error frequency in elongation factor Tu to 0.12 mistake per asparagine codon, while with lysine starvation in the same strain the error frequency per lysine codon was 0.008. The pattern of isoelectric point changes in the altered protein produced is consistent with third position misreading in the AAN codon group. This high level of mistranslation is not seen in streptomycin resistant (rpsL) strains or in most relA+ strains.