Severe ethanol stress inhibits yeast proteasome activity at moderate temperatures but not at low temperatures.

Since yeast research under laboratory conditions is usually conducted at 25-30°C (moderate temperature range), most of the findings on yeast physiology are based on analyses in this temperature range. Due to inefficiencies in cultivation and analysis, insufficient information is available on yeast physiology in the low-temperature range, although alcoholic beverage production is often conducted at relatively low temperatures (around 15°C). Recently, we reported that severe ethanol stress (10% v/v) inhibits proteasomal proteolysis in yeast cells under laboratory conditions at 28°C. In this study, proteasomal proteolysis at a low temperature (15°C) was evaluated using cycloheximide chase analysis of a short-lived protein (Gic2-3HA), an auxin-inducible degron system (Paf1-AID*-6FLAG), and Spe1-3HA, which is degraded ubiquitin-independently by the proteasome. At 15°C, proteasomal proteolysis was not inhibited under severe ethanol stress, and sufficient proteasomal activity was maintained. These results provide novel insights into the effects of low temperature and ethanol on yeast physiology.

Adaptability of wine yeast to ethanol-induced protein denaturation.

This year marks the 200th anniversary of the birth of Dr Louis Pasteur (1822-1895), who revealed that alcoholic fermentation is performed by yeast cells. Subsequently, details of the mechanisms of alcoholic fermentation and glycolysis in yeast cells have been elucidated. However, the mechanisms underlying the high tolerance and adaptability of yeast cells to ethanol are not yet fully understood. This review presents the response and adaptability of yeast cells to ethanol-induced protein denaturation. Herein, we describe the adverse effects of severe ethanol stress on intracellular proteins and the responses of yeast cells. Furthermore, recent findings on the acquired resistance of wine yeast cells to severe ethanol stress that causes protein denaturation are discussed, not only under laboratory conditions, but also during the fermentation process at 15°C to mimic the vinification process of white wine.

Acquired resistance to severe ethanol stress-induced inhibition of proteasomal proteolysis in Saccharomyces cerevisiae.

BACKGROUND: Although the budding yeast, Saccharomyces cerevisiae, produces ethanol via alcoholic fermentation, high-concentration ethanol is harmful to yeast cells. Severe ethanol stress (> 9% v/v) inhibits protein synthesis and increases the level of intracellular protein aggregates. However, its effect on proteolysis in yeast cells remains largely unknown. METHODS: We examined the effects of ethanol on proteasomal proteolysis in yeast cells through the cycloheximide-chase analysis of short-lived proteins. We also assayed protein degradation in the auxin-inducible degron system and the ubiquitin-independent degradation of Spe1 under ethanol stress conditions. RESULTS: We demonstrated that severe ethanol stress strongly inhibited the degradation of the short-lived proteins Rim101 and Gic2. Severe ethanol stress also inhibited protein degradation in the auxin-inducible degron system (Paf1-AID*-6FLAG) and the ubiquitin-independent degradation of Spe1. Proteasomal degradation of these proteins, which was inhibited by severe ethanol stress, resumed rapidly once the ethanol was removed. These results suggested that proteasomal proteolysis in yeast cells is reversibly inhibited by severe ethanol stress. Furthermore, yeast cells pretreated with mild ethanol stress (6% v/v) showed proteasomal proteolysis even with 10% (v/v) ethanol, indicating that yeast cells acquired resistance to proteasome inhibition caused by severe ethanol stress. However, yeast cells failed to acquire sufficient resistance to severe ethanol stress-induced proteasome inhibition when new protein synthesis was blocked with cycloheximide during pretreatment, or when Rpn4 was lost. CONCLUSIONS AND GENERAL SIGNIFICANCE: Our results provide novel insights into the adverse effects of severe ethanol stress on proteasomal proteolysis and ethanol adaptability in yeast.

Wine Yeast Cells Acquire Resistance to Severe Ethanol Stress and Suppress Insoluble Protein Accumulation during Alcoholic Fermentation.

Under laboratory conditions, acute 10% (vol/vol) ethanol stress causes protein denaturation and accumulation of insoluble proteins in yeast cells. However, yeast cells can acquire resistance to severe ethanol stress by pretreatment with mild ethanol stress (6% vol/vol) and mitigate insoluble protein accumulation under subsequent exposure to 10% (vol/vol) ethanol. On the other hand, protein quality control (PQC) of yeast cells during winemaking remains poorly understood. Ethanol concentrations in the grape must increase gradually, rather than acutely, to more than 10% (vol/vol) during the winemaking process. Gradual increases in ethanol evoke two possibilities for yeast PQC under high ethanol concentrations in the must: suppression of insoluble protein accumulation through the acquisition of resistance or the accumulation of denatured insoluble proteins. We examined these two possibilities by conducting alcoholic fermentation tests at 15°C that mimic white winemaking using synthetic grape must (SGM). The results obtained revealed the negligible accumulation of insoluble proteins in wine yeast cells throughout the fermentation process. Furthermore, wine yeast cells in fermenting SGM did not accumulate insoluble proteins when transferred to synthetic defined (SD) medium containing 10% (vol/vol) ethanol. Conversely, yeast cells cultured in SD medium accumulated insoluble proteins when transferred to fermented SGM containing 9.8% (vol/vol) ethanol. Thus, wine yeast cells acquire resistance to the cellular impact of severe ethanol stress during fermentation and mitigate the accumulation of insoluble proteins. This study provides novel insights into the PQC and robustness of wine yeast during winemaking. IMPORTANCE Winemaking is a dynamic and complex process in which ethanol concentrations gradually increase to reach >10% (vol/vol) through alcoholic fermentation. However, there is little information on protein damage in wine yeast during winemaking. We investigated the insoluble protein levels of wine yeast under laboratory conditions in SD medium and during fermentation in SGM. Under laboratory conditions, wine yeast cells, as well as laboratory strain cells, accumulated insoluble proteins under acute 10% (vol/vol) ethanol stress, and this accumulation was suppressed by pretreatment with 6% (vol/vol) ethanol. During the fermentation process, insoluble protein levels were maintained at low levels in wine yeast even when the SGM ethanol concentration exceeded 10% (vol/vol). These results indicate that the progression of wine yeast through fermentation in SGM results in stress tolerance, similar to the pretreatment of cells with mild ethanol stress. These findings further the understanding of yeast cell physiology during winemaking.