Use of PMA1 as a Housekeeping Biomarker for Assessment of Toxicant-Induced Stress in Saccharomyces cerevisiae

Abstract

The brewer's yeast Saccharomyces cerevisiae has emerged as a versatile and robust model system for laboratory use to study toxic effects of various substances. In this study, toxicant-induced stresses of pure compounds were investigated in Saccharomyces cerevisiae utilizing a destabilized version of the green fluorescent protein optimized for expression in yeast (yEGFP3) Conventionally, toxicologists have used bioassays based on rodent models to evaluate the toxic effects of chemical compounds and to study the mechanism of action of toxicants. However, scientific developments are required to keep in line with regulatory frameworks, such as existing EU guidelines for assessment of manufactured chemicals (67/548/EEC, 93/67/ EEC, and 83/571/EEC) and the EU regulatory framework for chemicals REACH 2003 (European Commission [http: //europa.eu.int/comm/enterprise/reach/index.htm]) concerning in part also existing chemicals. Scientific developments include the requirement for rapid and reliable high-throughput assays to evaluate more accurately and more mechanistically the potential hazards of large numbers of chemicals.The yeast Saccharomyces cerevisiae is a promising model for such assays because it is amenable to genetic studies and because of the vast amount of genomics knowledge, resources, and manipulative tools associated with this unicellular fungus. The high degree of homology of essential cellular organization and metabolism shared by S. cerevisiae and higher eukaryotes has enabled study of aspects of cellular toxicity and phenomena of relevance to human biology at the molecular level (5,30). Such research has offered many insights into the complex mechanisms underlying the sensing and response to toxicant stressors (13,14). The degree to which gene expression profiles are conserved upon toxic stress and the regulation of key pathway elements enabled the identification of human signal transduction homologues (2, 15). Although gene expression profiling is not (yet) suited for high-throughput screening, insights in terms of hierarchic clustering of genetic stress-related networks potentially provides the means to identify surrogate markers that can be used to construct detection systems and prediction models.PMA1, one of the most prominent housekeeping genes in S. cerevisiae, encodes the major plasma membrane H ϩ -ATPase (35) and is essential for viability. As a highly conserved member of the P-type ATPases, the H ϩ -ATPase is a single 100-kDa polypeptide. The electrogenic proton pump is the major source of cytosolic proton extrusion and generation of the proton motive force across the cellular membrane. The proton motive force is responsible for secondary active transport mechanisms for a variety of nutrients and is also involved in pH homeostasis. In being the major protein component of the plasma membrane (15 to 20% of total plasma membrane protein [1]), expression and activity of this proton pump are precisely regulated to match its numerous requirements (4,11,19,38).We have previously described the constr...

Saccharomyces cerevisiae and Caffeine Implications on the Eukaryotic Cell

“…The pleiotropic effect of caffeine on S. cerevisiae can be used to develop a model to study the toxic effects of various substances [ 30 ]. It was found that caffeine toxicity is enhanced in yeast cells following exposure to cigarette smoke and that yeast efflux transporters are targets of cigarette smoke chemicals, suggesting once more that associating caffeine-rich products with smoking is not recommended [ 31 ].…”

Yeast as a Humanized Model Organism for Biotransformation-Related Toxicity

“…Additionally, assays for general cytotoxicity have been developed using the promoter of the housekeeping plasma membrane ATPase gene PMA1 coupled to a reporter as biomarker [81]. Finally, yeast has been used as an indicator organism for the detection of the food toxin deoxynivalenol [82] and to examine the presence of toxicants in wastewater [83].…”

“…In several of the assays described above, ABC multidrug transporter encoding genes are deleted to increase the sensitivity of yeast to the toxic compound of interest [74,77,81,82]. Additionally, since yeast is a unicellular organism it lacks the different cell environments and structures of the various organs in mammals and, on a smaller scale, multicellular properties like gap junctions.…”

Engineering Bacteria for Bioremediation

“…al., 2006;Wu et. al., 2006), and though few studies have been developed for yeast, we can cite (Schmitt et. al., 2006) expressing EC20 with the aim of identifying microorganisms with increased capacity for binding heavy metal ions for use in bioremediation processes.…”