Induction of autophagy by second-fermentation yeasts during elaboration of sparkling wines

Abstract

Autophagy is a transport system mediated by vesicles, ubiquitous in eukaryotic cells, by which bulk cytoplasm is targeted to a lysosome or vacuole for degradation. In the yeast Saccharomyces cerevisiae, autophagy is triggered by nutritional stress conditions (e.g., carbon- or nitrogen-depleted medium). In this study we showed that there is induction of autophagy in second-fermentation yeasts during sparkling wine making. Two methods were employed to detect autophagy: a biochemical approach based on depletion of the protein acetaldehyde dehydrogenase Ald6p and a morphological strategy consisting of visualization of autophagic bodies and autophagosomes, which are intermediate vesicles in the autophagic process, by transmission electron microscopy. This study provides the first demonstration of autophagy in second-fermentation yeasts under enological conditions. The correlation between autophagy and yeast autolysis during sparkling wine production is discussed, and genetic engineering of autophagy-related genes in order to accelerate the aging steps in wine making is proposed.

FIG. 1.

Western blot analysis of protein extracts from strains BY4741 and ΔATG1 incubated in SD-N or YPD at 17°C. The membranes were probed with rabbit monoclonal anti-acetaldehyde dehydrogenase antibody.

FIG. 2.

Transmission electron micrographs of BY4741 (A), ΔATG1 (B), and EC1118 (C) cells after 8 h of incubation in SD-N containing 1 mM PMSF. In panel D arrowheads indicate the single membrane delimiting an autophagic body. Isolated autophagic bodies (arrows) were detected in EC1118 incubated for 8 h in SD-N without PMSF (E). No vesicles were detected in EC1118 vacuoles after 8 h of incubation in YPD containing 1 mM PMSF (F). AB, autophagic body; MIT, mitochondrion; N, nucleus; V, vacuole.

FIG. 3.

(A) Western blot of EC1118 extracts for 16 days of incubation at 17°C in Erlenmeyer flasks with base wine. The membranes were probed with rabbit monoclonal anti-acetaldehyde dehydrogenase antibody. (B) Sugar consumption in the same experiment. The error bars indicate standard deviations.

FIG. 4.

Transmission electron micrographs of EC1118 cells during the second fermentation of base wine. PMSF (1 mM) was added to the base wine 8 days after inoculation. After 8 h of incubation with the protease inhibitor, cells were removed and analyzed by TEM. The arrows indicate autophagic bodies in the vacuole. V, vacuole; N, nucleus; MIT, mitochondrion.

FIG. 5.

(A) Monitoring of a second-fermentation experiment in closed bottles with base wine and S. cerevisiae EC1118. The error bars indicate standard deviations. (B) Western blot of EC1118 protein extracts from the same experiment. The membranes were probed with rabbit monoclonal anti-acetaldehyde dehydrogenase antibody.

FIG. 6.

Transmission electron micrographs of EC1118 cells incubated for 10 days in a closed bottle with base wine. (A) Autophagic body. (B) Autophagosome. (C) Autophagosome precursor. (D and E) Enlarged images of the autophagosome and the autophagosome precursor. The arrows indicate the cup-shaped membrane, and the arrowheads indicate places where the cup-shaped membrane is fused to membranes. (F) Schematic representation of the autophagosome formation process according to the model of Kirisako et al. (18), shown for comparison. CYT, cytoplasm; V, vacuole; N, nucleus; MIT, mitochondrion.