Evaluation of the lower protein limit in the budding yeast Saccharomyces cerevisiae using TIPI-gTOW

Background

Identifying permissible limits of intracellular parameters such as protein expression provides important information for examining robustness. In this study, we used the TEV protease-mediated induction of protein instability (TIPI) in combination with the genetic Tug-of-War (gTOW) to develop a method to measure the lower limit of protein level. We first tested the feasibility of this method using ADE2 as a marker and then analyzed some cell cycle regulators to reveal genetic interactions.

Results

Using TIPI-gTOW, we successfully constructed a strain in which GFP-^TDegFAde2 was expressed at the lower limit, just sufficient to support cellular growth under the -Ade condition by accelerating degradation by TEV protease. We also succeeded in constructing a strain in which the minimal level of GFP-^TDegFCdc20 was expressed by TIPI-gTOW. Using this strain, we studied genetic interactions between cell cycle regulators and CDC20, and the result was highly consistent with the previously identified interactions. Comparison of the experimental data with predictions of a mathematical model revealed some interactions that were not implemented into the current model.

Conclusions

TIPI-gTOW is useful for estimating changes in the lower limit of a protein under different conditions, such as different genetic backgrounds and environments. TIPI-gTOW is also useful for analyzing genetic interactions of essential genes whose deletion mutants cannot be obtained.     

Cold-active acid beta-galactosidase activity of isolated psychrophilic-basidiomycetous yeast Guehomyces pullulans

In the present study, psychrophilic yeasts, which grow on lactose as a sole carbon source at low temperature and under acidic conditions, were isolated from soil from Hokkaido, Japan. The phenotypes and sequences of 28S rDNA of the isolated strains indicated a taxonomic affiliation to Guehomyces pullulans. The isolated strains were able to grow on lactose at below 5 degrees C, and showed cold-active acid beta-galactosidase activity even at 0 degrees C and pH 4.0 in the extracellular fractions. Moreover, K(m) of beta-galactosidase activity for lactose in the extracellular fraction from strain R1 was found to be 50.5 mM at 10 degrees C, and the activity could hydrolyze lactose in milk at 10 degrees C. The findings in this study indicate the possibility that the isolated strains produce novel acid beta-galactosidases that are able to hydrolyze lactose at low temperature.     

Prooxidant Action of Maltol: Role of Transition Metals in the Generation of Reactive Oxygen Species and Enhanced Formation of 8-hydroxy-2′-deoxyguanosine Formation in DNA

Maltol (3-hydroxy-2-methyl-4-pyrone) produced reactive oxygen species as a complex with transition metals. Maltol/iron complex inactivated aconitase the most sensitive enzyme to oxidative stress. The inactivation of aconitase was iron-dependent, and prevented by TEMPOL, a scavenger of reactive oxygen species, suggesting that the maltol/iron-mediated generation of superoxide anion is responsible for the inactivation of aconitase. Addition of maltol effectively enhanced the ascorbate/copper-mediated formation of 8-hydroxy-2′-deoxyguanosine in DNA. Oxidation of ascorbic acid by CuSO₄ was effectively stimulated by addition of maltol, and the enhanced oxidation rate was markedly inhibited by the addition of catalase and superoxide dismutase. These results suggest that maltol can stimulate the copper reduction coupled with the oxidation of ascorbate, resulting in the production of superoxide radical which in turn converts to hydrogen peroxide and hydroxyl radical. Cytotoxic effect of maltol can be explained by its prooxidant properties: maltol/transition metal complex generates reactive oxygen species causing the inactivation of aconitase and the production of hydroxyl radical causing the formation of DNA base adduct.