Optimized Mechanical Isolation of Mitochondria From Saccharomyces cerevisiae Preserving Atg32 for Quantitative...

Mitophagy is a highly conserved process among eukaryotic cells, playing a primordial role in mitochondrial quality control and overall cellular homeostasis. In Saccharomyces cerevisiae, Atg32 is the only identified mitophagy receptor localized to the mitochondrial outer membrane, making this yeast a particularly powerful model for molecular studies of mitophagy that require the isolation of intact mitochondria. However, traditional methods for isolating mitochondria from yeast often rely on enzymatic cell wall digestion and homogenization, which can compromise the stability of mitochondrial surface proteins such as Atg32. In this protocol, we describe an optimized mechanical approach for yeast cell disruption using glass beads in a cold, protease-inhibited buffer to preserve mitochondrial integrity and facilitate the detection of Atg32. Subsequent differential centrifugation and washing steps yield mitochondrial fractions suitable for downstream biochemical analyses. This workflow eliminates enzymatic digestion steps, reduces sample variability, and allows parallel processing of multiple strains or experimental conditions. Overall, this method offers a rapid, low-cost, and reproducible alternative for crude mitochondrial isolation, ensuring excellent preservation of Atg32 and broad compatibility with quantitative and comparative studies. Key features • Mechanical cell disruption using glass beads preserves mitochondrial integrity and enables reliable immunodetection of Atg32 without requiring enzymatic spheroplasting. • Rapid, low-cost, and highly reproducible workflow suitable for processing multiple yeast strains or experimental conditions in parallel. • Optimized cold, protease-inhibited lysis conditions minimize Atg32 degradation and improve detection sensitivity in mitochondrial fractions.