Mutations sensitizing yeast cells to the start inhibitor nalidixic acid
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J A Prendergast1, R A Singer, N Rowley
1Biochemistry Department, University of Alberta, Edmonton, Canada.
Abstract:
The regulatory step Start in the cell cycle of the budding yeast Saccharomyces cerevisiae is inhibited by nalidixic acid (Nal). To study this inhibition, mutations were identified that alter the sensitivity of yeast cells to Nal. Nal-sensitive mutations were sought because the inhibitory effects of Nal on wild-type cells are only transient, and wild-type cells naturally become refractory to Nal. Three complementation groups of Nal-sensitive mutations were found. Mutations in the first complementation group were shown to reside in the ARO7 gene, encoding chorismate mutase; tyrosine and phenylalanine synthesis was inhibited by Nal in these aro7 mutants, whereas wild-type chorismate mutase was unaffected, These aro7 alleles demonstrate 'recruitment', by mutation, of an innately indifferent protein to an inhibitor-sensitive form. The Nal-sensitive aro7 mutant cells were used to show that the resumption of Nal-inhibited nuclear activity and cell proliferation takes place while cytoplasmic Nal persists at concentrations inhibitory for the mutant chorismate mutase. Mutations in the second complementation group, nss2 (Nal-supersensitive), increased intracellular Nal concentrations, and may simply alter the permeability of cells to Nal. The third complementation group was found to be the ERG6 gene, previously suggested to encode the ergosterol biosynthetic enzyme sterol methyltransferase. Mutation or deletion of the ERG6 gene had little effect on the inhibition of Start by Nal, but prevented recovery from this inhibition. Mutation of ERG3, encoding another ergosterol biosynthetic enzyme, also caused Nal sensitivity, suggesting that plasma membrane sterol composition, and plasma membrane function, mediates recovery from Nal-mediated inhibition of Start.
Insights
Nalidixic acid (Nal) inhibits yeast cell cycle Start. Mutations in ARO7, NSS2, ERG6, and ERG3 genes reveal mechanisms of Nal sensitivity and recovery, involving chorismate mutase, cell permeability, and ergosterol biosynthesis.
Area of Science:
- Cell Biology
- Molecular Biology
- Genetics
Background:
- The cell cycle regulatory step 'Start' in Saccharomyces cerevisiae is inhibited by nalidixic acid (Nal).
- Wild-type yeast cells exhibit transient sensitivity to Nal, becoming refractory over time.
- Understanding Nal's inhibitory mechanisms and yeast's recovery pathways is crucial for cell cycle research.
Purpose of the Study:
- To identify and characterize mutations conferring nalidixic acid sensitivity in yeast.
- To elucidate the molecular basis of Nal inhibition and recovery from cell cycle arrest.
- To investigate the role of specific genes and metabolic pathways in mediating Nal sensitivity.
Main Methods:
- Screening for nalidixic acid-sensitive mutants in Saccharomyces cerevisiae.
- Complementation analysis to group mutations into distinct genetic loci.
- Biochemical assays to assess enzyme activity (chorismate mutase) and intracellular drug concentrations.
- Genetic analysis of ERG6 and ERG3 genes involved in ergosterol biosynthesis.
Main Results:
- Three complementation groups of Nal-sensitive mutations were identified: ARO7, NSS2, and ERG6.
- Mutations in ARO7 (chorismate mutase) render the enzyme sensitive to Nal, affecting tyrosine and phenylalanine synthesis.
- NSS2 mutations increase intracellular Nal concentrations, potentially affecting cell permeability.
- ERG6 and ERG3 mutations do not affect initial Nal inhibition but prevent recovery, implicating ergosterol in membrane function and recovery.
Conclusions:
- Nalidixic acid sensitivity can arise from direct enzyme inhibition (ARO7) or altered drug uptake (NSS2).
- Recovery from Nal-induced cell cycle arrest is dependent on ergosterol biosynthesis and plasma membrane integrity.
- These findings provide insights into the regulation of the cell cycle and drug resistance mechanisms in yeast.