The Ty1 transposition assay: a new short-term test for detection of carcinogens

An assay based on induction by carcinogens of Ty1 transposition in Saccharomyces cerevisiae is proposed. A tester strain was developed that contains a marked Ty1 element, which allows following the transposition in the genome as a whole and a mutation, which increases cellular permeability. Hypersensitivity to chemical agents, higher cell wall porosity and transformability with plasmid DNA evidenced an enhanced cellular permeability of the tester cells. The increased permeability resulted in higher sensitivity to carcinogens. The treatment with different laboratory carcinogens induced Ty1 transposition rates in the tester strain by a factor of 10 to 20, compared to the controls. The induction is not stress-generated by the cytotoxicity of carcinogens, since treatment with NaN3 at concentrations killing 50% of the cells did not increase the transposition rate. The increase of Ty1 transposition in tester cells is specific for active carcinogens and a positive response with procarcinogens was obtained only in presence of S9 mix. The Ty1 transposition test responded positively to a number of Ames-test or DEL-test negative carcinogens. The positive response of Ty1 test was statistically significant and verified in kinetics and concentration-dependent experiments. It is concluded that the Ty1 transposition test can be used, in addition to the Ames assay, as a short-term test for detection of carcinogens.     

Transposition of Saccharomyces cerevisiae Ty1 retrotransposon is activated by improper cryopreservation

Ty1 is a retrotransposon of the yeast Saccharomyces cerevisiae whose transposition at new locations in the host genome is activated by stress conditions, such as exposure to UV light, X-rays, nitrogen starvation. In this communication, we supply evidence that cooling for 2 h at +4 °C followed by freezing for 1 h at −10 °C and 16 h at −20 °C also increased Ty1 transposition. The mobility of Ty1 was induced by cooling at slow rates (3 °C/min) and the accumulation of trehalose inside cells or the cooling at high rates (100 °C/min) inhibited significantly the induction of the transposition. The freeze-induced Ty1 transposition did not occur in mitochondrial mutants (rho⁻) and in cells with disrupted SCO1 gene (Δsco1 cells) evidencing that the Ty1 transposition induced by cooling depends on the mitochondrial oxidative phosphorylation. We also found that the freeze induced Ty1 transposition is associated with increased synthesis and accumulation of superoxide anions (O⁻₂) into the cells. Accumulation of O⁻₂ and activation of Ty1 transposition were not observed after cooling of cells with compromised mitochondrial functions (rho⁻, Δsco1), or in cells pretreated with O⁻₂ scavengers. It is concluded that (i) elevated levels of reactive oxygen species (ROS) have a key role in activation the transposition of Ty1 retrotransposon in yeast cells undergoing freezing and (ii) given the deleterious effect of increased ROS levels on cells, special precautions should be taken to avoid ROS production and accumulation during cryopreservation procedures.     

Oltipraz-induced phase 2 enzyme response conserved in cells lacking mitochondrial DNA

Oltipraz, a member of a class of 1,2-dithiolethiones, is a potent phase 2 enzyme inducing agent used as a cancer chemopreventive. In this study, we investigated regulation of the phase 2 enzyme response and protection against endogenous oxidative stress in lymphoblastic leukemic parental CEM cells and cells lacking mitochondrial DNA (mtDNA) (rho0) by oltipraz. Glutathione (GSH) levels (total and mitochondrial) and glutathione S-transferase (GST) activity were significantly increased after pretreatment with oltipraz in both parental (rho+) and rho0 cells, and both cell lines were resistant to mitochondrial oxidation, loss of mitochondrial membrane potential, and cell death in response to the GSH depleting agent diethylmaleate. These results show that the phase 2 enzyme response, by enhancing GSH-dependent systems involved in xenobiotic metabolism, blocks endogenous oxidative stress and cell death, and that this response is intact in cells lacking mtDNA.     

LRRK2, but not pathogenic mutants,protects against H2O2 stress depending on mitochondrial function and endocytosis in a yeast model

Background

Mutations in LRRK2 are the most common genetic cause of Parkinson's disease (PD). Studies in the yeast Saccharomyces cerevisiae have provided valuable insights into the mechanisms of cellular dysfunction associated with the expression of faulty PD genes.

Methods

We developed a yeast model for full-length LRRK2 studies. We expressed wild-type (wt) LRRK2 and mutations and evaluated their role during oxidative stress conditions. The involvement of mitochondria was assessed by using rho-zero mutants and by evaluating reactive oxygen species (ROS) production and mitochondrial membrane potential by flow cytometry. The involvement of endocytosis was also studied by testing several endocytic mutants and by following the vacuolar delivery of the probe FM4-64.

Results

Expression of LRRK2 in yeast was associated to increased hydrogen peroxide resistance. This phenotype, which was dependent on mitochondrial function, was not observed for PD-mutants G2019S and R1441C or in the absence of the kinase activity and the WD40 repeat domain. Expression of the pathogenic mutants stimulated ROS production and increased mitochondrial membrane potential. For the PD-mutants, but not for wild-type LRRK2, endocytic defects were also observed. Additionally, several endocytic proteins were required for LRRK2-mediated protection against hydrogen peroxide.

Conclusions

Our results indicate that LRRK2 confers cellular protection during oxidative stress depending on mitochondrial function and endocytosis.

General significance

Both the loss of capacity of LRRK2 pathogenic mutants to protect against oxidative stress and their enhancement of dysfunction may be important for the development of PD during the aging process.     

Influence of a mitochondrial genetic defect on capacitative calcium entry and mitochondrial organization in the osteosarcoma cells

Effects of T8993G mutation in mitochondrial DNA (mtDNA), associated with neurogenical muscle weakness, ataxia and retinitis pigmentosa (NARP), on the cytoskeleton, mitochondrial network and calcium homeostasis in human osteosarcoma cells were investigated. In 98% NARP and rho(0) (lacking mtDNA) cells, the organization of the mitochondrial network and actin cytoskeleton was disturbed. Capacitative calcium entry (CCE) was practically independent of mitochondrial energy status in osteosarcoma cell lines. The significantly slower Ca(2+) influx rates observed in 98% NARP and rho(0), in comparison to parental cells, indicates that proper actin cytoskeletal organization is important for CCE in these cells.     

Mmm1p, a mitochondrial outer membrane protein, is connected to mitochondrial DNA (mtDNA) nucleoids and required for mtDNA stability

In the yeast Saccharomyces cerevisiae, mitochondria form a branched, tubular reticulum in the periphery of the cell. Mmm1p is required to maintain normal mitochondrial shape and in mmm1 mutants mitochondria form large, spherical organelles. To further explore Mmm1p function, we examined the localization of a Mmm1p-green fluorescent protein (GFP) fusion in living cells. We found that Mmm1p-GFP is located in small, punctate structures on the mitochondrial outer membrane, adjacent to a subset of matrix-localized mitochondrial DNA nucleoids. We also found that the temperature-sensitive mmm1-1 mutant was defective in transmission of mitochondrial DNA to daughter cells immediately after the shift to restrictive temperature. Normal mitochondrial nucleoid structure also collapsed at the nonpermissive temperature with similar kinetics. Moreover, we found that mitochondrial inner membrane structure is dramatically disorganized in mmm1 disruption strains. We propose that Mmm1p is part of a connection between the mitochondrial outer and inner membranes, anchoring mitochondrial DNA nucleoids in the matrix.     

Diaminotoluenes induce intrachromosomal recombination and free radicals in Saccharomyces cerevisiae

The carcinogenicity of aniline-based aromatic amines is poorly reflected by their activity in short-term mutagenicity assays such as the Salmonella typhimurium reverse mutation (Ames) assay. More information about the mechanism of action of such carcinogens is needed. Here we report the effects on DEL recombination in Saccharomyces cerevisiae of the carcinogen 2,4-diaminotoluene and its structural isomer 2,6-diaminotoluene, which is reported to be non-carcinogenic. Both compounds are detected as equally mutagenic in the Salmonella assay. In the absence of any external metabolizing system both compounds were recombinagenic in the DEL assay, with the carcinogen being a more potent inducer of deletions than the non-carcinogen. In the presence of Aroclor-induced rat liver S9, however, the carcinogen 2,4-diaminotoluene became a 2-fold more potent inducer of deletions, and the non-carcinogen 2,6-diaminotoluene was rendered less toxic and no induced recombination was observed. 2,4-Diaminotoluene is distinguished from its non-carcinogen analog in the DEL assay, therefore, on the basis of a preferential activation of the carcinogen in the presence of a rat liver microsomal metabolizing system. Free radical species are produced by several carcinogens and have been implicated in carcinogenesis. We further investigated whether exposure of yeast to either 2,4-diaminotoluene or 2,6-diaminotoluene resulted in a rise in intracellular free radical species. The effects of the free radical scavenger N-acetylcysteine on toxicity and recombination induced by the two compounds and intracellular oxidation of the free radical-sensitive reporter compound dichlorofluorescin diacetate were studied. Both 2,4- and 2,6-diaminotoluene produced free radical species in yeast, indicating that the reason for the differential activity of the compounds for induced deletions is not reflected in any difference in the production of free radical species.     

Protein overexport in a Saccharomyces cerevisiae mutant depends on mitochondrial genome integrity and function

The wild-type yeast Saccharomyces cerevisiae (S. cerevisiae) is able to export less than 1 percent of the protein to be secreted. The reasons for retention of most of the secretory proteins on the cell surface of S. cerevisiae are unknown. Recently, temperature-sensitive (ts) mutants of S. cerevisiae showing an oversecretion phenotype were isolated. In order to study the influence of the mitochondrial genome status on protein export in yeast cells, we have isolated several types of respiratory impaired mitochondrial mutants of either the parental S. cerevisiae strain or their derivative ts protein-overexporting mutants. In this paper we demonstrate by quantitative analyses of exported proteins and by SDS-PAGE analysis that protein overexport in ts mutants requires mitochondrial genome integrity and function.     

Musical chairs during mitophagy

Mitophagy, or the autophagic degradation of mitochondria, is thought to be important in mitochondrial quality control, and hence in cellular physiology. Defects in mitophagy correlate with late onset pathologies and aging. Here, we discuss recent results that shed light on the interrelationship between mitophagy and mitochondrial dynamics, based on proteomic analyses of protein dynamics in wild-type and mutant cells. These studies show that different mitochondrial matrix proteins undergo mitophagy at different rates, and that the rate differences are affected by mitochondrial dynamics. These results are consistent with models in which phase separation within the mitochondrial matrix leads to unequal segregation of proteins during mitochondrial fission. Repeated fusion and fission cycles may thus lead to “distillation” of components that are destined for degradation.     

Cellular iron utilization is regulated by putative siderophore transporter FgSit1 not by free iron transporter in Fusarium graminearum

This report investigated FgSit1, which encodes a putative ferrichrome transporter of Fusarium graminearum. The identity of the deduced amino acid sequence of FgSit1 with the amino acid sequence of ScArn1p, an FC-Fe(3+) transporter of Saccharomyces cerevisiae, was 51%; both the growth defect related to the Deltafet3Deltaarn1-4 strain of S. cerevisiae in an iron-depleted condition and the FC-Fe(3+) uptake activity were recovered upon the introduction of FgSit1 into the Deltafet3Deltaarn1-4 strain. Although ScArn1p was found in the late endosomal compartment in S. cerevisiae, FgSit1 was found on the plasma membrane in S. cerevisiae; when FgSit1 was expressed exogenously in S. cerevisiae, it showed greater FC-Fe(3+) uptake activity than did ScArn1p. Additionally, in F. graminearum FC-Fe(3+) uptake activity in the Deltafgsit1 strain was found to be one-fourth that of the wild-type. However, Fe(3+) uptake activity in the Deltafgsit1 strain was 5-fold higher than that of wild-type; the gene expression of FgFtr1, a putative iron transporter, was induced by the deletion of FgSit1, but was not induced by the deletion of FgSit2. Taken together, these results strongly suggest that FgSit1 encodes a putative FC-Fe(3+) transporter that mediates FC-Fe(3+) uptake using a different mechanism than ScArn1p and plays an important role in the regulation of cellular iron availability in F. graminearum.     

Mitochondrial genome content is regulated during nematode development

Growth and development rely on the mitochondrial respiratory chain (MRC) as the major source of ATP. We measured the mitochondrial DNA (mtDNA) copy number of each of the Caenorhabditis elegans developmental stages. Embryos, L1, L2, and L3 larvae all have approximately 25,000 copies of maternally derived mtDNA. The copy number increases fivefold in L4 larvae and a further sixfold in adult hermaphrodites, but only twofold in adult males. The majority of mtDNA in adult worms is germline associated, and germline-deficient mutants show markedly reduced mtDNA contents. With sperm-deficient or oocyte-deficient mutants, we confirm that mtDNA amplification is primarily associated with oocyte production. When mtDNA replication is inhibited, a quantitative and homogeneous arrest as L3 larvae occurs. Thus, mtDNA amplification is a necessary component of normal development and its regulation may involve an energy-sensing decision or checkpoint that can be invoked when mitochondrial energy generation is impaired.     

Analyzing the suppression of respiratory defects in the yeast model of human mitochondrial tRNA diseases

The respiratory defects associated with mutations in human mitochondrial tRNA genes can be mimicked in yeast, which is the only organism easily amenable to mitochondrial transformation. This approach has shown that overexpression of several nuclear genes coding for factors involved in mitochondrial protein synthesis can alleviate the respiratory defects both in yeast and in human cells.     

Biogenesis of yeast mitochondrial cytochrome c: a unique relationship to the TOM machinery

The import of cytochrome c into the mitochondrial intermembrane space is not understood at a mechanistic level. While the precursor apocytochrome c can insert into protein-free lipid bilayers, the purified translocase of the outer membrane (TOM) complex supports the translocation of apocytochrome c into proteoliposomes. We report an in organello analysis of cytochrome c import into yeast mitochondria from wild-type cells and different mutants cells, each defective in one of the seven Tom proteins. The import of cytochrome c is not affected by removal of the receptor Tom20 or Tom70. Moreover, neither the transfer protein Tom5 nor the assembly factors Tom6 and Tom7 are needed for import of cytochrome c. When the general import pore (GIP)-protein Tom40 is blocked, the import of cytochrome c is moderately affected. Mitochondria lacking the central receptor and organizing protein Tom22 contain greatly reduced levels of cytochrome c. We conclude that up to two components of the TOM complex, Tom22 and possibly the GIP, are involved in the biogenesis of cytochrome c.