Abstract
Stress in the endoplasmic reticulum caused by tunicamycin, dithiothreitol, and azole-class antifungal drugs can induce nonapoptotic cell death in yeasts that can be blocked by the action of calcineurin (Cn), a Ca(2+)-dependent serine/threonine protein phosphatase. To identify additional factors that regulate nonapoptotic cell death in yeast, a collection of gene knock-out mutants was screened for mutants exhibiting altered survival rates. The screen revealed an endocytic protein (Ede1) that can function upstream of Ca(2+)/calmodulin-dependent protein kinase 2 (Cmk2) to suppress cell death in parallel to Cn. The screen also revealed the vacuolar H(+)-ATPase (V-ATPase), which acidifies the lysosome-like vacuole. The V-ATPase performed its death-promoting functions very soon after imposition of the stress and was not required for later stages of the cell death program. Cn did not inhibit V-ATPase activities but did block vacuole membrane permeabilization (VMP), which occurred at late stages of the cell death program. All of the other nondying mutants identified in the screens blocked steps before VMP. These findings suggest that VMP is the lethal event in dying yeast cells and that fungi may employ a mechanism of cell death similar to the necrosis-like cell death of degenerating neurons.
Highlights
Living cells utilize several mechanisms to commit suicide when stressed
These findings suggest that vacuole membrane permeabilization (VMP) is the lethal event in dying yeast cells and that fungi may employ a mechanism of cell death similar to the necrosis-like cell death of degenerating neurons
The high-affinity Ca2ϩ influx system (HACS)-deficient mutants cch1, mid1, and ecm7 exhibited higher frequencies of cell death in both conditions (83 and 96%, 90 and 96%, and 46 and 96% death, respectively), which was closely mimicked by cultures of cnb1 cmk2 double mutants that simultaneously lack both targets of HACS
Summary
Living cells utilize several mechanisms to commit suicide when stressed. Results: We find that dying yeast cells utilize the V-ATPase to release toxic materials into the cytoplasm. All of the other nondying mutants identified in the screens blocked steps before VMP These findings suggest that VMP is the lethal event in dying yeast cells and that fungi may employ a mechanism of cell death similar to the necrosis-like cell death of degenerating neurons. The genetic disruption of HACS, calmodulin, or calcineurin (Cn) or the pharmacological inhibition of Cn with either FK506 or cyclosporine was not harmful to yeast growth or mating in ordinary circumstances Deficiencies in this calcium signaling pathway were completely lethal during prolonged exposures to mating pheromones. The findings suggest that HACS, [Ca2ϩ]i elevation, calmodulin, and Cn constitute a signaling pathway that actively suppresses a pheromone-inducible cell death program through regulation of protein phosphorylation. We find V-ATPase activity to be necessary for death of yeast cells similar to the requirement for V-ATPase activity in the necrosis-like death of degenerating neurons [5] and the chloroquine-induced death of cerebellar granule neurons [30]
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