Abstract

Crabtree effect, defined as the propensity of yeast to prioritize fermentation over respiration in high-glucose environments, is a classic metabolic adaptation. However, the limitations of conventional mitochondrial isolation methods have hindered in-depth investigation of the effect’s suborganellar impact. To directly probe this process at the organelle level, we developed an innovative antibody-based immunoaffinity capture strategy using magnetic zirconium-based metal–organic framework materials named Fe3O4/UiO-66-NH2@Avidin@TOM70. This approach capitalizes on the high surface area of magnetic metal–organic framework and the stable biotin–avidin interaction to achieve high-specificity enrichment of intact yeast mitochondria. Compared to traditional sucrose density gradient centrifugation and tagged magnetic beads, the developed method demonstrated faster separation, superior mitochondrial purity, and better structural integrity. Comprehensive metabolomics and lipidomics analyses revealed that the Crabtree effect triggers extensive mitochondrial metabolic reprogramming, rather than merely functional inhibition. These findings demonstrate that the acetyl-CoA metabolic flux has undergone a fundamental rewiring in addition to the expected downregulation of TCA cycle. Evidence of this remodeling includes a reduction in acetic acid levels and a significant distribution of lipid synthesis, which is conducive to the production of triglycerides and phosphatidic acid rather than major membrane phospholipids such as phosphatidylcholine and phosphatidylethanolamine. Meanwhile, the perturbations in the biosynthesis of mitochondrial-related amino acids were observed. This research not only provides a powerful tool for organelle isolation but also offers novel, subcellular-resolved insights into how the Crabtree effect globally reconfigures mitochondrial metabolism and lipid networks to support the fermenting lifestyle of yeast.