Mutant Yeast Strains Research Articles

Searching for the Function of Ecm14, a Pseudopeptidase, Using a Synthetic Lethal Assay

Many enzyme families contain both catalytically active and inactive members. Although these inactive enzyme‐homologs (pseudoenzymes) share significant similarity with their active counterparts, key active site modifications seem to suggest a loss of traditional catalytic activity. The human M14 metallocarboxypeptidase (MCP) family is one such enzyme family that contains several active and inactive members. The baker's yeast Saccharomyces cerevisiae contains only one M14 MCP member, Ecm14, which is produced by a non‐essential gene in the yeast genome. We show through a phylogenetic analysis that this gene is present throughout the ascomycete lineage of fungi. Although Ecm14 is very similar to human CPA1 (a catalytically active member of the M14 MCP family), multiple key modifications in Ecm14 seem to indicate a loss of traditional peptidase‐like activity. A key technique for elucidating the function of non‐essential genes, including those producing pseudoenzyme products, is the genome‐wide synthetic lethal screen. Biochemical pathways typically involve multiple gene components and many of these gene players provide redundancy in the event that other genes experience deleterious mutations. The synthetic lethal screen is used to identify novel mutant yeast strains whose survival is dependent on a particular gene of interest. Colonies that contain a putative synthetically lethal gene pair are kept alive and identified by harboring a color‐reporting plasmid that complements the deletion of the endogenous gene of interest. Here we demonstrate the insertion of the wild‐type ECM14 gene into this integral pSLS1 plasmid and the determination of gene insert orientation using restriction endonucleases. Additionally, we demonstrate the preparation of knockout yeast strains using drug resistant deletion cassettes, the transformation of these yeast with the above plasmid, and finally, genome‐wide mutagenesis attempts using the chemical mutagen ethyl methanesulfonate. Identification of synthetic lethal genes will be presented, as a step toward a better understanding of the biological role of Ecm14.Support or Funding InformationGraduate Student Grants in Aid of Research to RCM and Faculty Research Grants to PJL from the Office of Research & Creative Scholarship, Andrews University.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

SUMOylation of the nuclear pore complex basket is involved in sensing cellular stresses.

ABSTRACTThe nuclear pore complex (NPC) is the major conduit for nucleocytoplasmic transport and serves as a platform for gene regulation and DNA repair. Several nucleoporins undergo ubiquitylation and SUMOylation, and these modifications play an important role in nuclear pore dynamics and plasticity. Here, we perform a detailed analysis of these post-translational modifications of yeast nuclear basket proteins under normal growth conditions as well as upon cellular stresses, with a focus on SUMOylation. We find that the balance between the dynamics of SUMOylation and deSUMOylation of Nup60 and Nup2 at the NPC differs substantially, particularly in G1 and S phase. While Nup60 is the unique target of genotoxic stress within the nuclear basket that probably belongs to the SUMO-mediated DNA damage response pathway, both Nup2 and Nup60 show a dramatic increase in SUMOylation upon osmotic stress, with Nup2 SUMOylation being enhanced in Nup60 SUMO-deficient mutant yeast strains. Taken together, our data reveal that there are several levels of crosstalk between nucleoporins, and that the post-translational modifications of the NPC serve in sensing cellular stress signals.

Heavy metal hypertolerant eukaryotic aldehyde dehydrogenase isolated from metal contaminated soil by metatranscriptomics approach

Constant addition of heavy metal pollutants in soil resulting from anthropogenic activities can prove detrimental to both macro and micro life forms inhabiting the ecosystem. The potential functional roles of eukaryotic microbes in such environment were explored in this study by metatranscriptomics approach. Sized eukaryotic cDNA libraries, library A (<0.5 kb), library B (0.5–1.0 kb), and library C (>1 kb) were constructed from the soil RNA and screened for copper (Cu) tolerance genes by using copper sensitive yeast mutant strain cup1Δ. Screening of the cDNA libraries yielded different clones capable of growing in Cu amended medium. In the present investigation, one of the transcripts PLCc38 from the library C was characterized and tested for its ability to tolerate different heavy metals by using metal sensitive yeast mutants. Sequence analysis PLCc38 showed homology with aldehyde dehydrogenase (ALDH) and capable of tolerating high concentrations of Cu (150–1000 μM). Aldeyde dehydrogenases are stress response enzymes capable of eliminating toxic levels of aldehydes generated due to abiotic environmental stresses. The cDNA PLCc38 also provided tolerance to wide range of Cd (40–100 μM), Zn (10–13 mM) and Co (2–50 mM) concentrations. Oxidative stress tolerance potential of PLCc38 was also confirmed in presence of different concentrations of H2O2. This study proves that PLCc38 is a potent gene associated with metal tolerance which could be used to revegetate heavy metal polluted soil or as a biomarker for detection of metal contamination.

Isolation of yeast Ca2+ signal transduction inhibitors from the Early Cretaceous Burmese amber

Two kinds of biologically active compounds were isolated from the MeOH extract of the Early Cretaceous Burmese amber [99 million years ago (Ma)], which is older than the K-Pg boundary (65 Ma). These compounds had inhibitory activity against the hypersensitive mutant yeast strain (zds1∆ erg3∆ pdr1∆ pdr3∆) with respect to Ca2+ signal transduction. They were identified as 16,17-bisnordehydroabietic acid (1) and 16,17-bisnorcallitrisic acid (2), respectively, on the basis of spectral analyses including HREIMS, 1D, and 2D NMR. Both have faint growth restoring activities around the clear inhibition zone against the mutant yeast on the 0.31–0.16 μg/spot. This is the first report of direct structural elucidation of 1 and 2 and the biologically active compounds derived from Burmese amber.

Biological activities of Chromolena odorata (L.) King and Robinson (Asteraceae) collected from Sabah, Malaysia as protein phosphatase type-1 inhibitor

Chromolena Odorata has been traditionally used as wound healer in local community. The present study investigated the anti-kinase and anti-phosphatases activities on methanolic C. odorata extract. Mutant yeast strains used are MKK1P386, MKK1P386_MSG5, PAY704-1 and PAY700-4. Bioassay guided fractionation of C. odorata revealed positive activities of hexane, ethyl acetate and chloroform partitions. Column chromatography of all partitionates later confirmed fraction F2 from chloroform extract had most favorable activity with inhibitory zone ranged between 7±0.0 mm until 15±0.0 mm. Kinetic analysis including maximum enzyme velocity (Vm) and Michealis-Menten constant (Km) were evaluated and compared for both normal and inhibited reactions. Enzyme activity with DiFMUP as substrate showed fraction F2 act as PP1 enzyme inhibitor with the Km value 0.60 mM and Vm value 200 mM/min as compared to the normal enzymatic reaction. Results provided unveil the potential of C. odorata as an effective therapeutic agent.

Exploring Novel Functions of the Small GTPase Ypt1p under Heat-Shock by Characterizing a Temperature-Sensitive Mutant Yeast Strain, ypt1-G80D.

In our previous study, we found that Ypt1p, a Rab family small GTPase protein, exhibits a stress-driven structural and functional switch from a GTPase to a molecular chaperone, and mediates thermo tolerance in Saccharomyces cerevisiae. In the current study, we focused on the temperature-sensitive ypt1-G80D mutant, and found that the mutant cells are highly sensitive to heat-shock, due to a deficiency in the chaperone function of Ypt1pG80D. This defect results from an inability of the protein to form high molecular weight polymers, even though it retains almost normal GTPase function. The heat-stress sensitivity of ypt1-G80D cells was partially recovered by treatment with 4-phenylbutyric acid, a chemical chaperone. These findings indicate that loss of the chaperone function of Ypt1pG80D underlies the heat sensitivity of ypt1-G80D cells. We also compared the proteomes of YPT1 (wild-type) and ypt1-G80D cells to investigate Ypt1p-controlled proteins under heat-stress conditions. Our findings suggest that Ypt1p controls an abundance of proteins involved in metabolism, protein synthesis, cellular energy generation, stress response, and DNA regulation. Finally, we suggest that Ypt1p essentially regulates fundamental cellular processes under heat-stress conditions by acting as a molecular chaperone.

OsPHT1;3 Mediates Uptake, Translocation, and Remobilization of Phosphate under Extremely Low Phosphate Regimes

Plant roots rely on inorganic orthophosphate (Pi) transporters to acquire soluble Pi from soil solutions that exists at micromolar levels in natural ecosystems. Here, we functionally characterized a rice (Oryza sativa) Pi transporter, Os Phosphate Transporter-1;3 (OsPHT1;3), that mediates Pi uptake, translocation, and remobilization. OsPHT1;3 was directly regulated by Os Phosphate Starvation Response-2 and, in response to Pi starvation, showed enhanced expression in young leaf blades and shoot basal regions and even more so in roots and old leaf blades. OsPHT1;3 was able to complement a yeast mutant strain defective in five Pi transporters and mediate Pi influx in Xenopus laevis oocytes. Overexpression of OsPHT1;3 led to increased Pi concentration both in roots and shoots. However, unlike that reported for other known OsPHT1 members that facilitate Pi uptake at relatively higher Pi levels, mutation of OsPHT1;3 impaired Pi uptake and root-to-shoot Pi translocation only when external Pi concentration was below 5 μm Moreover, in basal nodes, the expression of OsPHT1;3 was restricted to the phloem of regular vascular bundles and enlarged vascular bundles. An isotope labeling experiment with 32P showed that ospht1;3 mutant lines were impaired in remobilization of Pi from source to sink leaves. Furthermore, overexpression and mutation of OsPHT1;3 led to reciprocal alteration in the expression of OsPHT1;2 and several other OsPHT1 genes. Yeast-two-hybrid, bimolecular fluorescence complementation, and coimmunoprecipitation assays all demonstrated a physical interaction between OsPHT1;3 and OsPHT1;2. Taken together, our results indicate that OsPHT1;3 acts as a crucial factor for Pi acquisition, root-to-shoot Pi translocation, and redistribution of phosphorus in plants growing in environments with extremely low Pi levels.

Cadmium and arsenic responses in the ectomycorrhizal fungus Laccaria bicolor: glutathione metabolism and its role in metal(loid) homeostasis.

Ectomycorrhizal fungi play an important role in protecting their host plant from metal(loid) stresses by synthesizing various thiol rich compounds like metallothioneins and glutathione. We investigated the effect of cadmium (Cd) and arsenic (As) stress with a specific interest on glutathione (GSH) in the ectomycorrhizal fungus Laccaria bicolor. The total GSH levels inside the cell were significantly increased with increase in external metal(loid) stress. An analysis of the transcript levels of genes responsible for GSH synthesis, γ-glutamylcysteine synthetase (Lbγ-GCS) and glutathione synthetase (LbGS), using qPCR revealed that expression of both genes increased as a function of external metal(loid) concentration. The enzyme activity of both Lbγ-GCS and LbGS were increased with increase in external Cd and As concentration. Further, the functional role of Lbγ-GCS and LbGS genes in response to Cd and As stress was studied using their respective yeast mutant strains gsh1 Δ and gsh2 Δ . The mutant strains successfully expressed the two genes resulting in wild-type phenotype restoration of Cd and As tolerance. From these results, it was concluded that GSH act as a core component in the mycorrhizal defence system under Cd and As stress for metal(loid) homeostasis and detoxification.

New polyketides, paralactonic acids A–E produced by Paraconiothyrium sp. SW-B-1, an endophytic fungus associated with a seaweed, Chondrus ocellatus Holmes

Five polyketides, paralactonic acids A-E (1–5) were isolated from Paraconiothyrium sp. SW-B-1, an endophytic fungus isolated from the seaweed, Chondrus ocellatus Holmes. Their structures were determined by various spectroscopic methods, predominantly by 2D NMR spectroscopic analyses. The absolute configurations of compounds 1–5 were determined by comparison of the experimental and calculated ECD spectra. Compound 5 showed moderate antibacterial activity and restored the growth of a mutant yeast strain inhibited by hyperactivated Ca2+-signaling.

Functional Identification of Salt-Stress-Related Genes Using the FOX Hunting System from Ipomoea pes-caprae.

Ipomoea pes-caprae is a seashore halophytic plant and is therefore a good model for studying the molecular mechanisms underlying salt and stress tolerance in plant research. Here, we performed Full-length cDNA Over-eXpressor (FOX) gene hunting with a functional screening of a cDNA library using a salt-sensitive yeast mutant strain to isolate the salt-stress-related genes of I. pes-caprae (IpSR genes). The library was screened for genes that complemented the salt defect of yeast mutant AXT3 and could grow in the presence of 75 mM NaCl. We obtained 38 candidate salt-stress-related full-length cDNA clones from the I. pes-caprae cDNA library. The genes are predicted to encode proteins involved in water deficit, reactive oxygen species (ROS) scavenging, cellular vesicle trafficking, metabolic enzymes, and signal transduction factors. When combined with the quantitative reverse transcription-polymerase chain reaction (qRT-PCR) analyses, several potential functional salt-tolerance-related genes were emphasized. This approach provides a rapid assay system for the large-scale screening of I. pes-caprae genes involved in the salt stress response and supports the identification of genes responsible for the molecular mechanisms of salt tolerance.

Transcriptome analysis revealed pivotal transporters involved in the reduction of cadmium accumulation in pak choi (Brassica chinensis L.) by exogenous hydrogen-rich water

Hydrogen-rich water (HRW) has been widely used in research on plant resistance to Cd. However, the underlying molecular mechanism of HRW in ameliorating cadmium stress in vegetables is largely unknown. In this study, the RNA-sequencing analyses were used to characterize the role of HRW in the alleviation of Cd toxicity in Chinese cabbage seedlings. Based on the obtained results, two genes encoding metal ionic transporters, BcIRT1 and BcZIP2 were ultimately selected out. Then, a systematic validation of the metal ion transport function of these two ZIP-encoding genes of pak choi were performed via a yeast transformation system. The results showed that BcIRT1 and BcZIP2 increased the sensitivity of different yeast mutant strains to relative metal ionic stresses and facilitated the accumulation of metal ions (Cd2+, Mn2+, Zn2+, and Fe2+) in yeast; thus, it suggests that BcIRT1 and BcZIP2 probably have the ability to transport Cd2+, Mn2+, Zn2+ and Fe2+ in pak choi. The time-course and concentration-dependent expression profiles of BcIRT1 and BcZIP2 showed that as time with HRW increased, the effectiveness of the repression on the expression of BcIRT1 and BcZIP2 increased, and as the seedlings were exposed to increased Cd concentrations, the inhibition of BcIRT1 and BcZIP2 by HRW was also increased. Over all, these findings provide new insights into the genome-wide transcriptome profiles in pak choi and show that HRW reduced Cd uptake probably through inhibiting the expression of transporters related to Cd absorption, BcIRT1 and BcZIP2.

Black sesame pigment extract from sesame dregs by subcritical CO2: Extraction optimization, composition analysis, binding copper and antioxidant protection

Black sesame pigment (BSP) is a natural product with good bioactivity. The massive accumulation of sesame dregs (SDs) from sesame seed processing provides a rich source of BSP. This study investigates the activity of BSP derived from SDs. A BSP yield of 3.58 ± 0.08% was achieved via extraction with subcritical CO2 under optimal conditions. BSP effectively bound Cu in vitro at a pH of 7.0 (R > 90%). Furthermore, binding activity was observed in Saccharomyces cerevisiae (43.78% reduction in intracellular Cu, BSP at 200 μg/mL). In addition, BSP conferred antioxidant protection in mutant yeast strains lacking superoxide dismutase and glutathione synthase by decreasing lipid peroxidation and reactive oxygen species (ROS). Superoxide dismutase and glutathione synthase protect against ROS by decreasing lipid peroxidation conferred by BSP. For decreasing intracellular ROS, only glutathione synthase seemed to be involved.

Using both qualitative and quantitative data in parameter identification for systems biology models

In systems biology, qualitative data are often generated, but rarely used to parameterize models. We demonstrate an approach in which qualitative and quantitative data can be combined for parameter identification. In this approach, qualitative data are converted into inequality constraints imposed on the outputs of the model. These inequalities are used along with quantitative data points to construct a single scalar objective function that accounts for both datasets. To illustrate the approach, we estimate parameters for a simple model describing Raf activation. We then apply the technique to a more elaborate model characterizing cell cycle regulation in yeast. We incorporate both quantitative time courses (561 data points) and qualitative phenotypes of 119 mutant yeast strains (1647 inequalities) to perform automated identification of 153 model parameters. We quantify parameter uncertainty using a profile likelihood approach. Our results indicate the value of combining qualitative and quantitative data to parameterize systems biology models.

The mechanisms of fructose 1,6 bisphosphatase degradation in methylotrophic yeasts Pichia pastoris

Aim. The study of the mechanisms of fructose-1,6-bisphosphatase degradation in methylotrophic yeasts Pichia pastoris. Methods. Methods of determination the specific activity of fructose-1,6-bisphosphatase in the wild type and mutant strains of methylotrophic yeast P. pastoris after shifting cells from the medium with methanol into the medium with glucose were used. The study of fructose-1,6-bisphosphatase protein degradetion was performed by Western blot analysis. Results. The changes of the specific activity of fructose-1,6-bisphosphatase in the wild type strain GS200, the strain with the deletion of the GSS1 hexose sensor gene and strain defected in autophagy pathway SMD1163 of P. pastoris in short-term and long-term induction with methanol, and with or without the addition of the MG132 (proteasome degradation inhibitor) was investigated. Degradation of fructose‑1,6‑bisphosphatase by the Western blot analysis in GS200, SMD1163 and Δgss1 strains was studied. Conclusions. It was shown that the duration of cell incubation on methanol has no particular effect on the inactivation of the enzyme. The effect of the proteasome inhibitor MG132 was insignificant. Catabolic inactivation of cytosolic and peroxisomal enzymes is damaged in the Δgss1 mutant as glucose signaling is impaired. Fructose-1,6-bisphosphatase degrades by a vacuolar pathway, regardless of the duration of methanol induction, which correlates with the activity data of this enzyme. Keywords: fructose-1,6-bisphosphatase, yeasts, Pichia pastoris, methanol, autophagy.

Cell Lysate Microarray for Mapping the Network of Genetic Regulators for Histone Marks

Proteins, as the major executer for cell progresses and functions, its abundance and the level of post-translational modifications, are tightly monitored by regulators. Genetic perturbation could help us to understand the relationships between genes and protein functions. Herein, to explore the impact of the genome-wide interruption on certain protein, we developed a cell lysate microarray on kilo-conditions (CLICK) with 4837 knockout (YKO) and 322 temperature-sensitive (ts) mutant strains of yeast (Saccharomyces cerevisiae). Taking histone marks as examples, a general workflow was established for the global identification of upstream regulators. Through a single CLICK array test, we obtained a series of regulators for H3K4me3, which covers most of the known regulators in S. cerevisiae We also noted that several group of proteins are involved in negatively regulation of H3K4me3. Further, we discovered that Cab4p and Cab5p, two key enzymes of CoA biosynthesis, play central roles in histone acylation. Because of its general applicability, CLICK array could be easily adopted to rapid and global identification of upstream protein/enzyme(s) that regulate/modify the level of a protein or the posttranslational modification of a non-histone protein.

Complete Native Stable Isotope Labeling by Amino Acids of Saccharomyces cerevisiae for Global Proteomic Analysis.

Knowledge about the functions of individual proteins on a system-wide level is crucial to fully understand molecular mechanisms underlying cellular processes. A considerable part of the proteome across all organisms is still poorly characterized. Mass spectrometry is an efficient technology for the global study of proteins. One of the most prominent methods for accurate proteome-wide comparative quantification is stable isotope labeling by amino acids in cell culture (SILAC). However, application of SILAC to prototrophic organisms such as Saccharomyces cerevisiae, also known as baker’s yeast, is compromised since they are able to synthesize all amino acids on their own. Here, we describe an advanced strategy, termed 2nSILAC, that allows for in vivo labeling of prototrophic baker’s yeast using heavy arginine and lysine under fermentable and respiratory growth conditions, making it a suitable tool for the global study of protein functions. This generic 2nSILAC strategy allows for directly using and systematically screening yeast mutant strain collections available to the scientific community. We exemplarily demonstrate its high potential by analyzing the effects of mitochondrial gene deletions in mitochondrial fractions using quantitative mass spectrometry revealing the role of Coi1 for the assembly of cytochrome c oxidase (respiratory chain complex IV).

Characterization of a non-nudix pyrophosphatase points to interplay between flavin and NAD(H) homeostasis in Saccharomyces cerevisiae.

The flavin cofactors FMN and FAD are required for a wide variety of biological processes, however, little is known about their metabolism. Here, we report the cloning and biochemical characterization of the Saccharomyces cerevisiae pyrophosphatase Fpy1p. Genetic and functional studies suggest that Fpy1p may play a key role in flavin metabolism and is the first-reported non-Nudix superfamily enzyme to display FAD pyrophosphatase activity. Characterization of mutant yeast strains found that deletion of fpy1 counteracts the adverse effects that are caused by deletion of flx1, a known mitochondrial FAD transporter. We show that Fpy1p is capable of hydrolyzing FAD, NAD(H), and ADP-ribose. The enzymatic activity of Fpy1p is dependent upon the presence of K+ and divalent metal cations, with similar kinetic parameters to those that have been reported for Nudix FAD pyrophosphatases. In addition, we report that the deletion of fpy1 intensifies the FMN-dependence of null mutants of the riboflavin kinase Fmn1p, demonstrate that fpy1 mutation abolishes the decreased fitness resulting from the deletion of the flx1 ORF, and offer a possible mechanism for the genetic interplay between fpy1, flx1 and fmn1.

Conserved functions of Arabidopsis mitochondrial late-acting maturation factors in the trafficking of iron‑sulfur clusters

Numerous proteins require iron‑sulfur (Fe-S) clusters as cofactors for their function. Their biogenesis is a multi-step process occurring in the cytosol and mitochondria of all eukaryotes and additionally in plastids of photosynthetic eukaryotes. A basic model of Fe-S protein maturation in mitochondria has been obtained based on studies achieved in mammals and yeast, yet some molecular details, especially of the late steps, still require investigation. In particular, the late-acting biogenesis factors in plant mitochondria are poorly understood. In this study, we expressed the factors belonging to NFU, BOLA, SUFA/ISCA and IBA57 families in the respective yeast mutant strains. Expression of the Arabidopsis mitochondrial orthologs was usually sufficient to rescue the growth defects observed on specific media and/or to restore the abundance or activity of the defective Fe-S or lipoic acid-dependent enzymes. These data demonstrate that the plant mitochondrial counterparts, including duplicated isoforms, likely retained their ancestral functions. In contrast, the SUFA1 and IBA57.2 plastidial isoforms cannot rescue the lysine and glutamate auxotrophies of the respective isa1-isa2Δ and iba57Δ strains or of the isa1-isa2-iba57Δ triple mutant when expressed in combination. This suggests a specialization of the yeast mitochondrial and plant plastidial factors in these late steps of Fe-S protein biogenesis, possibly reflecting substrate-specific interactions in these different compartments.

Cloning and functional characterization of long-chain acyl-CoA synthetase 1 from the mesocarp of African oil palm (Elaeis guineensis Jacq.)

African oil palm (Elaeis guineensis Jacq.) is the highest-yielding oil crop in the world. Recent studies have shown the importance of long-chain acyl-CoA synthetases (LACSs) in lipid biosynthesis and degradation, but few studies have focused on oil palm. In this work, we found that transcription of EgLACS1was positively correlated with lipid accumulation in the oil palm mesocarp. In order to clarify the role of EgLACS1 on lipid metabolism, three variants of the EgLACS1 gene were cloned from oil palm and designated EgLACS1-V1, EgLACS1-V2, and EgLACS1-V3. Sequence analysis revealed that the three variant proteins contained several highly conserved motifs, shared important sequence similarities with other known LACS1s, and demonstrated that EgLACS1-V1, EgLACS1-V2 and EgLACS1-V3 were 666-, 664- and 601-aminoacid proteins, respectively. All of the three variant genes encodingEgLACS1 were expressed and tested for their functionality in an acyl-coenzyme-A synthetase- (ACS-) deficient mutant yeast strain YB525. Only EgLACS1-V1 and EgLACS1-V2 could rescue the growth deficiency (an inability to grow on a fatty acid auxotrophic medium) of YB525 in complementation tests. Using a fluorescent fatty acid analogue confirmed that EgLACS1-V1 and EgLACS1-V2 facilitated exogenous fatty acid uptake. EgLACS1 overexpression in yeast reduced fatty acid content. All these results suggested that not only did both the EgLACS1-V1 and EgLACS1-V2 enzymes exhibit acyl-coenzyme-A activities, but that they were also involved in the transfer of fatty acid-coenzyme A. They may also take part in the storage of lipid during fruit development in oil palm. The characterization of EgLACS1 will provide a molecular basis for the study of acyl-coenzyme A synthetase-mediated lipid synthesis and degradation in oil palm.

Metal induction of two metallothionein genes in the ectomycorrhizal fungus Suillus himalayensis and their role in metal tolerance.

Metallothioneins (MTs) are small proteins with highly conserved cysteine residues and are involved in metal homeostasis and metal detoxification. Two metallothionein genes ShMT1 and ShMT2 from the ectomycorrhizal fungus Suillus himalayensis were characterised for their potential role in heavy metal detoxification. The response of these MTs to the exogenous concentrations of copper and cadmium was studied by qPCR analysis. The exogenous copper but not the cadmium at the tested concentrations induced the expression of the MT genes. The functional role of ShMTs was validated by expressing the two genes through functional complementation in yeast mutant strain cup1Δ (copper-sensitive), ycf1Δ (cadmium- sensitive) and zrc1Δ (zinc-sensitive). The mutant strain successfully expressed the two genes resulting in wild-type phenotype restoration of copper, cadmium and zinc tolerance. The present study shows that the ectomycorrhizal fungus S. himalayensis encodes two metallothionein genes (ShMT1 and ShMT2) which are more inducible by copper than cadmium and could play an important role in their detoxification.