Mutations sensitizing yeast cells to the start inhibitor nalidixic acid

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.