Generation and characterization of single and multigene Arabidopsis thaliana mutants in LSU1-4 (RESPONSE TO LOW SULFUR) genes.

In Arabidopsis thaliana, there are four members of the LSU (RESPONSE TO LOW SULFUR) gene family which are tandemly located on chromosomes 3 (LSU1 and LSU3) and 5 (LSU2 and LSU4). The LSU proteins are small, with coiled-coil structures, and they are able to form homo- and heterodimers. LSUs are involved in plant responses to environmental challenges, such as sulfur deficiency, and plant immune responses. Assessment of the role and function of these proteins was challenging due to the absence of deletion mutants. Our work fulfills this gap through the construction of a set of LSU deletion mutants (single, double, triple, and quadruple) by CRISPR/Cas9 technology. The genomic deletion regions in the obtained lines were mapped and the level of expression of each LSUs was assayed in each mutant. All lines were viable and capable of seed production. Their growth and development were compared at several different stages with the wild-type. No significant and consistent differences in seedlings' growth and plant development were observed in the optimal conditions. In sulfur deficiency, the roots of 12-day-old wild-type seedlings exhibited increased length compared to optimal conditions; however, this difference in root length was not observed in the majority of lsu-KO mutants.

Control of ABA Signaling and Crosstalk with Other Hormones by the Selective Degradation of Pathway Components.

A rapid and appropriate genetic and metabolic acclimation, which is crucial for plants' survival in a changing environment, is maintained due to the coordinated action of plant hormones and cellular degradation mechanisms influencing proteostasis. The plant hormone abscisic acid (ABA) rapidly accumulates in plants in response to environmental stress and plays a pivotal role in the reaction to various stimuli. Increasing evidence demonstrates a significant role of autophagy in controlling ABA signaling. This field has been extensively investigated and new discoveries are constantly being provided. We present updated information on the components of the ABA signaling pathway, particularly on transcription factors modified by different E3 ligases. Then, we focus on the role of selective autophagy in ABA pathway control and review novel evidence on the involvement of autophagy in different parts of the ABA signaling pathway that are important for crosstalk with other hormones, particularly cytokinins and brassinosteroids.

Partial Inhibition of Calcineurin Activity by Rcn2 as a Potential Remedy for Vps13 Deficiency.

Regulation of calcineurin, a Ca2+/calmodulin-regulated phosphatase, is important for the nervous system, and its abnormal activity is associated with various pathologies, including neurodegenerative disorders. In yeast cells lacking the VPS13 gene (vps13Δ), a model of VPS13-linked neurological diseases, we recently demonstrated that calcineurin is activated, and its downregulation reduces the negative effects associated with vps13Δ mutation. Here, we show that overexpression of the RCN2 gene, which encodes a negative regulator of calcineurin, is beneficial for vps13Δ cells. We studied the molecular mechanism underlying this effect through site-directed mutagenesis of RCN2. The interaction of the resulting Rcn2 variants with a MAPK kinase, Slt2, and subunits of calcineurin was tested. We show that Rcn2 binds preferentially to Cmp2, one of two alternative catalytic subunits of calcineurin, and partially inhibits calcineurin. Rcn2 ability to bind to and reduce the activity of calcineurin was important for the suppression. The binding of Rcn2 to Cmp2 requires two motifs in Rcn2: the previously characterized C-terminal motif and a new N-terminal motif that was discovered in this study. Altogether, our findings can help to better understand calcineurin regulation and to develop new therapeutic strategies against neurodegenerative diseases based on modulation of the activity of selected calcineurin isoforms.

The sulfur metabolism regulator MetR is a global regulator controlling phytochrome-dependent light responses in Aspergillus nidulans.

Phytochrome-dependent light signaling has been studied in several fungi. In Aspergillus nidulans light-stimulated phytochrome activates the high-osmolarity glycerol (HOG) signaling pathway and thereby controls the expression of a large number of genes, many of which are related to stress responses. In a genome-wide expression analysis in A. nidulans we found that phytochrome, fphA, is under strict expression control of the central regulator of the sulfur-starvation response, MetR. This transcriptional regulator is required for the expression of genes involved in inorganic sulfur assimilation. In the presence of organic sulfur, MetR is probably ubiquitinated and possibly degraded and the transcription of sulfur-assimilation genes, e.g., sulfate permease, is turned off. The expression analysis described here revealed, however, that MetR additionally controls the expression of hundreds of genes, many of which are required for secondary metabolite production. We also show that metR mutation phenocopies fphA deletion, and five other histidine-hybrid kinases are down-regulated in the metR1 mutant. Furthermore, we found that light and phytochrome regulate the expression of at least three carbon-sulfur hydrolases. This work is a further step towards understanding the interplay between light sensing and metabolic pathways.

Similar but Not Identical-Binding Properties of LSU (Response to Low Sulfur) Proteins From Arabidopsis thaliana.

Members of the plant-specific LSU (RESPONSE TO LOW SULFUR) family are strongly induced during sulfur starvation. The molecular functions of these proteins are unknown; however, they were identified as important stress-related hubs in several studies. In Arabidopsis thaliana, there are four members of the LSU family (LSU1-4). These proteins are small (approximately 100 amino acids), with coiled-coil structures. In this work, we investigated interactions between different monomers of LSU1-4. Differences in homo- and heterodimer formation were observed. Our structural models of LSU1-4 homo- and heterodimers were in agreement with our experimental observations and may help understand their binding properties. LSU proteins are involved in multiple protein-protein interactions, with the literature suggesting they can integrate abiotic and biotic stress responses. Previously, LSU partners were identified using the yeast two hybrid approach, therefore we sought to determine proteins co-purifying with LSU family members using protein extracts isolated from plants ectopically expressing TAP-tagged LSU1-4 constructs. These experiments revealed 46 new candidates for LSU partners. We tested four of them (and two other proteins, CAT2 and NBR1) for interaction with LSU1-4 by other methods. Binding of all six proteins with LSU1-4 was confirmed by Bimolecular Fluorescence Complementation, while only three of them were interacting with LSUs in yeast-two-hybrid. Additionally, we conducted network analysis of LSU interactome and revealed novel clues for the possible cellular function of these proteins.

Lack of G1/S control destabilizes the yeast genome via replication stress-induced DSBs and illegitimate recombination.

The protein Swi6 in Saccharomyces cerevisiae is a cofactor in two complexes that regulate the transcription of the genes controlling the G1/S transition. It also ensures proper oxidative and cell wall stress responses. Previously, we found that Swi6 was crucial for the survival of genotoxic stress. Here, we show that a lack of Swi6 causes replication stress leading to double-strand break (DSB) formation, inefficient DNA repair and DNA content alterations, resulting in high cell mortality. Comparative genome hybridization experiments revealed that there was a random genome rearrangement in swi6Δ cells, whereas in diploid swi6Δ/swi6Δ cells, chromosome V is duplicated. SWI4 and PAB1, which are located on chromosome V and are known multicopy suppressors of swi6Δ phenotypes, partially reverse swi6Δ genome instability when overexpressed. Another gene on chromosome V, RAD51, also supports swi6Δ survival, but at a high cost; Rad51-dependent illegitimate recombination in swi6Δ cells appears to connect DSBs, leading to genome rearrangement and preventing cell death.This article has an associated First Person interview with the first author of the paper.

Molecular characterization of central cytoplasmic loop in Aspergillus nidulans AstA transporter.

AstA (alternative sulfate transporter) belongs to a large, but poorly characterized, Dal5 family of allantoate permeases of the Major Facilitator Superfamily. The astA gene has been cloned from an IAM 2006 Japanese strain of Aspergillus nidulans by complementation of a sulfate permease-deficient mutant. In this study we show that conserved lysine residues in Central Cytoplasmic Loop (CCL) of the AstA protein may participate in anion selectivity, and control kinetic properties of the AstA transporter. A three-dimensional model containing four clustered lysine residues was created, showing a novel substrate-interacting structure in Major Facilitator Superfamily transporters. The assimilation constant (Kτ) of wild type AstA protein is 85 µM, while Vmax/mg of DW of AstA is twice that of the main sulfate transporter SB per mg of dry weight (DW) of mycelium (1.53 vs. 0.85 nmol/min, respectively). Amino acid substitutions in CCL did not abolish sulfate uptake, but affected its kinetic parameters. Mutants affected in the lysine residues forming the postulated sulfate-interacting pocket in AstA were able to grow and uptake sulfate, indicating that CCL is not crucial for sulfate transportation. However, these mutants exhibited altered values of Kτ and Vmax, suggesting that CCL is involved in control of the transporter activity.

The budding yeast orthologue of Parkinson's disease-associated DJ-1 is a multi-stress response protein protecting cells against toxic glycolytic products.

Saccharomyces cerevisiae Hsp31p is a DJ-1/ThiJ/PfpI family protein that was previously shown to be important for survival in the stationary phase of growth and under oxidative stress. Recently, it was identified as a chaperone or as glutathione-independent glyoxalase. To elucidate the role played by this protein in budding yeast cells, we investigated its involvement in the protection against diverse environmental stresses. Our study revealed that HSP31 gene expression is controlled by multiple transcription factors, including Yap1p, Cad1p, Msn2p, Msn4p, Haa1p and Hsf1p. These transcription factors mediate the HSP31 promoter responses to oxidative, osmotic and thermal stresses, to potentially toxic products of glycolysis, such as methylglyoxal and acetic acid, and to the diauxic shift. We also demonstrated that the absence of the HSP31 gene sensitizes cells to these stressors. Overproduction of Hsp31p and its homologue Hsp32p rescued the sensitivity of glo1Δ cells to methylglyoxal. Hsp31p also reversed the increased sensitivity of the ald6Δ strain to acetic acid. Since Hsp31p glyoxalase III coexists in S. cerevisiae cells with thousand-fold more potent glyoxalase I/II system, its biological purpose requires substantiation. We postulate that S. cerevisiae Hsp31p may have broader substrate specificity than previously proposed and is able to eliminate various toxic products of glycolysis. Alternatively, Hsp31p might be effective under high concentration of exogenous methylglyoxal present in some natural environmental niches populated by budding yeast, when glyoxalase I/II system capacity is saturated.

Phosphatidylinositol-3-phosphate regulates response of cells to proteotoxic stress.

Human Nedd4 ubiquitin ligase, or its variants, inhibit yeast cell growth by disturbing the actin cytoskeleton organization and dynamics, and lead to an increase in levels of ubiquitinated proteins. In a screen for multicopy suppressors which rescue growth of yeast cells producing Nedd4 ligase with an inactive WW4 domain (Nedd4w4), we identified a fragment of ATG2 gene encoding part of the Atg2 core autophagy protein. Expression of the Atg2-C1 fragment (aa 1074-1447) improved growth, actin cytoskeleton organization, but did not significantly change the levels of ubiquitinated proteins in these cells. The GFP-Atg2-C1 protein in Nedd4w4-producing cells primarily localized to a single defined structure adjacent to the vacuole, surrounded by an actin filament ring, containing Hsp42 and Hsp104 chaperones. This localization was not affected in several atg deletion mutants, suggesting that it might be distinct from the phagophore assembly site (PAS). However, deletion of ATG18 encoding a phosphatidylinositol-3-phosphate (PI3P)-binding protein affected the morphology of the GFP-Atg2-C1 structure while deletion of ATG14 encoding a subunit of PI3 kinase suppressed toxicity of Nedd4w4 independently of GFP-Atg2-C1. Further analysis of the Atg2-C1 revealed that it contains an APT1 domain of previously uncharacterized function. Most importantly, we showed that this domain is able to bind phosphatidylinositol phosphates, especially PI3P, which is abundant in the PAS and endosomes. Together our results suggest that human Nedd4 ubiquitinates proteins in yeast and causes proteotoxic stress and, with some Atg proteins, leads to formation of a perivacuolar structure, which may be involved in sequestration, aggregation or degradation of proteins.

The Aspergillus nidulans metZ gene encodes a transcription factor involved in regulation of sulfur metabolism in this fungus and other Eurotiales.

In Aspergillus nidulans, expression of sulfur metabolism genes is activated by the MetR transcription factor containing a basic region and leucine zipper domain (bZIP). Here we identified and characterized MetZ, a new transcriptional regulator in A. nidulans and other Eurotiales. It contains a bZIP domain similar to the corresponding region in MetR and this similarity suggests that MetZ could potentially complement the MetR deficiency. The metR and metZ genes are interrupted by unusually long introns. Transcription of metZ, unlike that of metR, is controlled by the sulfur metabolite repression system (SMR) dependent on the MetR protein. Overexpression of metZ from a MetR-independent promoter in a ΔmetR background activates transcription of genes encoding sulfate permease, homocysteine synthase and methionine permease, partially complementing the phenotype of the ΔmetR mutation. Thus, MetZ appears to be a second transcription factor involved in regulation of sulfur metabolism genes.

Regulatory mutations affecting sulfur metabolism induce environmental stress response in Aspergillus nidulans.

Mutations in the cysB, sconB and sconC genes affect sulfur metabolism in Aspergillus nidulans in different ways. The cysB mutation blocks synthesis of cysteine by the main pathway and leads to a shortage of this amino acid. The sconB and sconC mutations affect subunits of the SCF ubiquitin ligase complex, which inactivates the MetR transcription factor in the presence of an excess of cysteine. In effect, both cysB and scon mutations lead to permanent derepression of MetR-dependent genes. We compared transcriptomes of these three mutants with that of a wild type strain finding altered expression of a few hundred genes belonging to various functional categories. Besides those involved in sulfur metabolism, many up-regulated genes are related to stress responses including heat shock and osmotic stress. However, only the scon strains are more resistant to exogenous stress agents than the wild type strain while cysB is more sensitive. The two-component signal transduction system is a functional category, which is most enriched among genes up-regulated in the cysB, sconB and sconC mutants. A large group of up-regulated genes are involved in carbohydrate and energy metabolism, including genes coding for enzymes of trehalose and glycerol synthesis. The altered expression of these genes is accompanied by changes in sugar and polyol accumulation in conidia of the mutants. Genes encoding enzymes of the glyoxylate bypass and the GABA shunt are also up-regulated along with genes coding for enzymes of alcohol fermentation. Among the down-regulated genes the most numerous are those encoding membrane proteins and enzymes involved in secondary metabolism, including the penicillin biosynthesis cluster.

Human hAtg2A protein expressed in yeast is recruited to preautophagosomal structure but does not complement autophagy defects of atg2Δ strain.

Yeast Atg2, an autophagy-related protein, is highly conserved in other fungi and has two homologues in humans, one of which is hAtg2A encoded by the hATG2A/KIAA0404 gene. Region of homology between Atg2 and hAtg2A proteins comprises the C-terminal domain. We used yeast atg2D strain to express the GFP-KIAA0404 gene, its fragment or fusions with yeast ATG2, and study their effects on autophagy. The GFP-hAtg2A protein localized to punctate structures, some of which colocalized with Ape1-RFP-marked preautophagosomal structure (PAS), but it did not restore autophagy in atg2Δ cells. N-terminal fragment of Atg2 and N-terminal fragment of hAtg2A were sufficient for PAS recruitment but were not sufficient to function in autophagy. Neither a fusion of the N-terminal fragment of hAtg2A with C-terminal domain of Atg2 nor a reciprocal fusion were functional in autophagy. hAtg2A, in contrast to yeast Atg2, did not show interaction with the yeast autophagy protein Atg9 but both Atg2 proteins showed interaction with Atg18, a phospholipid-binding protein, in two-hybrid system. Moreover, deletion of ATG18 abrogated PAS recruitment of hAtg2A. Our results show that human hAtg2A can not function in autophagy in yeast, however, it is recruited to the PAS, possibly due to the interaction with Atg18.

Aspergillus nidulans genes encoding reverse transsulfuration enzymes belong to homocysteine regulon.

Homocysteine is an intermediate in methionine synthesis in Aspergillus nidulans, but it can also be converted to cysteine by the reverse transsulfuration pathway involving cystathionine beta-synthase (CBS) and cystathionine gamma-lyase (CGL). Because homocysteine is toxic to the cell at high concentrations, this pathway also functions as a means of removal of its excess. We found that the transcription of the mecA and mecB genes encoding CBS and CGL was upregulated by excess of homocysteine as well as by shortage of cysteine. Homocysteine induced transcription of both genes when added to the growth medium or overproduced in a regulatory mutant. The derepressing effect of cysteine shortage was observed in some mutants and in the wild-type strain during sulfur starvation. An increase in the level of mecA or mecB transcript roughly parallel with the elevation of the respective enzyme activity. On the basis of the mode of mecA and mecB regulation by homocysteine, these genes may be classified in a group of genes upregulated directly or indirectly by this amino acid. We call this group of genes the "homocysteine regulon".

Multiple fungal enzymes possess cysteine synthase activity in vitro.

We present evidence that there are at least three Aspergillus nidulans enzymes which catalyze in vitro the reaction of O-acetylserine (OAS) with sulfide forming cysteine. This activity is shared by cysteine synthase (CS) encoded by the cysB gene, homocysteine synthase encoded by cysD and by at least one more enzyme. Moreover, arginine, histidine or proline starvation leads to derepression of CS activity even in the cysB,cysD double mutant strains, while neither cysB nor cysD gene transcription is derepressed by amino acid starvation. Using a cpcA mutant, we show that starvation-inducible CS activity is under control of cross-pathway regulation. We identify CysF as a putative CS in A. nidulans. However, cysF gene transcription is not elevated by amino acid starvation. Therefore, it seems that there exists yet another enzyme, thus far unidentified, which possesses CS activity. Using mutants impaired during various steps of cysteine synthesis we prove that the cysB-encoded enzyme is the only CS of physiological importance in the studied fungus. Similar results were obtained with Schizosaccharomyces pombe mutant strains impaired in cysteine synthesis, indicating that the presence of multiple enzymes with in vitro CS activity may be a common feature of many fungal species.

Sulfate transport in Aspergillus nidulans: a novel gene encoding alternative sulfate transporter.

In Aspergillus nidulans sulfate is taken up by sulfate permease encoded by the sB gene. A unique tight auxotrophic mutant with an impaired promoter region of the sulfate permease gene, sB1(pr), was isolated. Three suppressor genes were cloned by complementation of this mutation. One of them, described here, is the astA gene (alternative sulfate transporter) derived from a genomic library of the Japanese A. nidulans IAM 2006 strain. In the reference strain of Glasgow origin the astA gene was found to be a pseudogene having several nucleotide deletions in ORF. The gene encodes a novel type of sulfate transporter which is distinct from other known sulfate permeases forming the SulP family. The putative ASTA protein belongs to an extensive and poorly characterized Dal5 allantoate permease family of fungal organic anion transporters. We have shown that ASTA is a physiological sulfate transporter. We also report cloning and characterization of the sB gene in this work. Both genes, sB and astA, are regulated at the transcriptional level by sulfur metabolite repression (SMR).

Two Aspergillus nidulans genes encoding methylenetetrahydrofolate reductases are up-regulated by homocysteine.

Methylenetetrahydrofolate reductase (MTHFR) catalyzes the reduction of 5,10-methylenetetrahydrofolate to 5-methyltetrahydrofolate, a co-substrate in the synthesis of methionine from homocysteine. We have cloned and characterized two Aspergillus nidulans genes encoding MTHFRs: metA and metF. Mutations in either gene result in methionine requirement; the metA-encoded enzyme is responsible for only 10-15% of total MTHFR activity. These two enzymes belong to different classes of MTHFRs. Mutations in metA but not in the metF gene are suppressed by mutations resulting in enhancement of homocysteine synthesis. The expression of both genes is up-regulated by homocysteine.

The Aspergillus nidulans metR gene encodes a bZIP protein which activates transcription of sulphur metabolism genes.

The identification, isolation and characterization of a new Aspergillus nidulans positive-acting gene metR, which encodes a transcriptional activator of sulphur metabolism, is reported. metR mutants are tight auxotrophs requiring methionine or homocysteine for growth. Mutations in the metR gene are epistatic to mutations in the negative-acting sulphur regulatory scon genes. The metR coding sequence is interrupted by a single intron of 492 bp which is unusually long for fungi. Aspergillus nidulans METR is a member of bZIP family of DNA-binding proteins. The bZIP domains of METR and the Neurospora crassa CYS3 transcriptional activator of sulphur genes are highly similar. Although Neurospora cys-3 gene does not substitute for the metR function, a chimeric metR gene with a cys-3 bZIP domain is able to transform the DeltametR mutant to methionine prototrophy. This indicates that METR recognizes the same regulatory sequence as CYS3. The metR gene is not essential, as deletion mutants are viable and have similar phenotype as point mutants. In contrast to the Neurospora cys-3, transcription of the metR gene was found to be regulated neither by METR protein nor by sulphur source. Transcription of metR gene is derepressed in the sconB2 mutant. Transcription of genes encoding sulphate permease, homocysteine synthase, cysteine synthase, ATP-sulphurylase, and sulphur controller--sconB is strongly regulated by the metR gene product and depends on the character of the metR mutation and sulphur supplementation.

Sulphur amino acid synthesis in Schizosaccharomyces pombe represents a specific variant of sulphur metabolism in fungi.

Schizosaccharomyces pombe, in contrast to Saccharomyces cerevisiae and Aspergillus nidulans, lacks cystathionine beta-synthase and cystathionine gamma-lyase, two enzymes in the pathway from methionine to cysteine. As a consequence, methionine cannot serve as an efficient sulphur source for the fungus and does not bring about repression of sulphur assimilation, which is under control of the cysteine-mediated sulphur metabolite repression system. This system operates at the transcriptional level, as was shown for the homocysteine synthase encoding gene. Our results corroborate the growing evidence that cysteine is the major low-molecular-weight effector in the regulation of sulphur metabolism in bacteria, fungi and plants.