A Mitochondria-Dependent Pathway Mediates the Apoptosis of GSE-Induced Yeast

Grapefruit seed extract (GSE), which has powerful anti-fungal activity, can induce apoptosis in S. cerevisiae. The yeast cells underwent apoptosis as determined by testing for apoptotic markers of DNA cleavage and typical chromatin condensation by Terminal Deoxynucleotidyl Transferase–mediated dUTP Nick End Labeling (TUNEL) and 4,6′-diaminidino-2-phenylindole (DAPI) staining and electron microscopy. The changes of ΔΨmt (mitochondrial transmembrane potential) and ROS (reactive oxygen species) indicated that the mitochondria took part in the apoptotic process. Changes in this process detected by metabonomics and proteomics revealed that the yeast cells tenaciously resisted adversity. Proteins related to redox, cellular structure, membrane, energy and DNA repair were significantly increased. In this study, the relative changes in the levels of proteins and metabolites showed the tenacious resistance of yeast cells. However, GSE induced apoptosis in the yeast cells by destruction of the mitochondrial 60 S ribosomal protein, L14-A, and prevented the conversion of pantothenic acid to coenzyme A (CoA). The relationship between the proteins and metabolites was analyzed by orthogonal projections to latent structures (OPLS). We found that the changes of the metabolites and the protein changes had relevant consistency

<i>S. cerevisiae</i> intracellular ROS.

<p>A–D, S. cerevisiae treated with 0.13 mg/mL of GSE for 0 h, 1 h, 2 h, and 3 h.</p

Representative silver-stained 2-D gel of total cellular extracts from yeast cells treated with GSE.

<p>The spots that were altered upon treatment are represented by numbers (1–26).</p

<i>S. cerevisiae</i> mitochondrial membrane potential (DYmt).

<p>A–G, S. cerevisiae treated with 0.13 mg/mL of GSE for 0, 0.5, 1, 1.5, 2, 2.5, and 3 h.</p

1H NMR spectra of a typical cell extract.

<p>1H NMR spectra of a typical cell extract.</p

DAPI- and TUNEL-stained <i>S. cerevisiae</i> nuclei (10×60).

<p>A, E, control group; B, C, D, S. cerevisiae treated with 0.13 mg/mL of GSE for 3 h; A–C yeast cells stained with DAPI; D and E yeast cells stained with TUNEL.</p