Enhancing Freezing Stress Tolerance through Regulation of the Ubiquitin–Proteasome System in Saccharomyces cerevisiae

Abstract

The baking industry is experiencing significant growth, primarily due to the widespread adoption of frozen dough baking. However, this process can negatively impact the fermentation ability of yeast, as freezing can induce stress in yeast cells. This study reports the molecular interplay between the ubiquitin–proteasome system and freezing stress tolerance in the yeast Saccharomyces cerevisiae. Using the proteasome inhibitor MG132, we first screened mutants with enhanced freezing stress tolerance. Three mutants showed elevated activity of the intracellular proteasome, particularly trypsin-like activity (more than threefold) and reduced sensitivity to MG132 inhibition of chymotrypsin-like activity (less than 0.125-fold). Genomic analysis of these mutants revealed mutations in the ROX1 gene, a heme-dependent repressor of hypoxic genes. Importantly, the ROX1 deletion strain displayed slightly improved freezing stress tolerance (about 1.5-fold). Comprehensive transcription analysis identified the ANB1 gene as a potential downstream target of Rox1. Overexpression of ANB1 enhanced freezing stress tolerance (about 1.5-fold) with increased the proteasome’s activity, indicating that Rox1 contributes to changes in the proteasome’s activity and freezing stress tolerance through the function of Anb1. The present data provide new insights into the mechanisms of freezing stress tolerance and help us improve the baking of frozen dough to produce higher-quality bread.