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In budding yeast, reactive oxygen species induce both RAS-dependent and RAS-independent cell cycle-specific arrest
1Dipartimento di Biotecnologie e Bioscienze, Università degli Studi di Milano-Bicocca, Milan, Italy.
Abstract:
The role of mild oxidative stresses elicited by diethylmaleate (DEM)-induced glutathione depletion in the progression of the yeast cell cycle has been investigated. We found that different wild-type strains are sensitive to oxidative stresses induced by similar DEM doses: approximately 1 mM on YPD plates, 5-10 mM in shaken flasks. At lower doses, DEM caused a transient decrease in growth rate, largely because of a decreased G1-to-S transition. Treatment with higher DEM doses leads to complete growth arrest, with most cells found in the unbudded G1 phase of the cell cycle. DEM treatment resulted in transcriptional induction of stress-responsive element (STRE)-controlled genes and was relieved by treatment with the antioxidant N-acetyl cysteine. Reciprocal shift experiments with cdc25 and cdc28 mutants showed that the major cell cycle arrest point was located in the Start area, at or near the CDC25-mediated step, before the step mediated by the CDC28 cyclin-dependent kinase. The DEM-induced G1 arrest requires a properly regulated RAS pathway and can be bypassed by overexpressing the G1-specific cyclin CLN2. However, cells with either a deregulated RAS pathway or overexpressing CLN2 failed to grow and arrested as budded cells, indicating that a second DEM-sensitive cell cycle step exists.
Insights
Mild oxidative stress from diethylmaleate (DEM) impacts yeast cell cycle progression. DEM causes growth arrest at G1, requiring a functional RAS pathway and CLN2 for proper cell division.
Area of Science:
- Cell Biology
- Biochemistry
- Yeast Genetics
Background:
- Oxidative stress is a critical factor influencing cellular processes.
- Glutathione depletion is a key mechanism by which oxidative stress impacts cells.
- The yeast cell cycle is a well-established model for studying fundamental cell division processes.
Purpose of the Study:
- To investigate the effects of diethylmaleate (DEM)-induced glutathione depletion on the yeast cell cycle.
- To identify the specific cell cycle phases affected by mild oxidative stress.
- To elucidate the molecular pathways involved in the oxidative stress response during the cell cycle.
Main Methods:
- Yeast strains were treated with varying doses of DEM to induce oxidative stress.
- Cell cycle progression was monitored using microscopy and growth assays.
- Transcriptional changes were analyzed by examining stress-responsive element (STRE)-controlled genes.
- Genetic analysis involving cdc25, cdc28, RAS pathway mutants, and CLN2 overexpression was performed.
Main Results:
- DEM treatment induced sensitivity in wild-type yeast strains, causing growth rate reduction and cell cycle arrest.
- Lower DEM doses primarily affected the G1-to-S transition, while higher doses led to G1 arrest.
- DEM induced transcriptional activation of stress-responsive genes, which was reversed by N-acetyl cysteine.
- The G1 arrest was dependent on the RAS pathway and CLN2, but deregulation or overexpression led to a secondary arrest in budded cells.
Conclusions:
- Mild oxidative stress, through glutathione depletion, significantly disrupts the yeast cell cycle, primarily at the G1 phase.
- The RAS pathway and CLN2 play crucial roles in regulating the response to DEM-induced oxidative stress.
- A secondary, distinct cell cycle checkpoint sensitive to DEM exists, highlighting the complexity of oxidative stress responses.