Figure 1. Workflow of the drug screen assay in reconstituted yeast mitochondria.

The yeast S. cerevisiae constitutes a versatile system that can be reconstituted with different components of the MCUC machinery (e.g. MCU and EMRE and additionally MCUb, MICU1 and/or MICU2, as represented with dashed lines). When D‐Lactate (D‐Lac) is supplied to mitochondria as the energy source, it provides a bioenergetic shunt pathway that minimizes the detection of false‐positive hits. This drug screen platform allows the quantification of mitochondrial Ca²⁺ uptake kinetics based on mitochondria‐targeted‐aequorin luminescence emitted at 469 nm. MCUC modulators are accurately identified based on their effects on mitochondrial Ca²⁺ uptake kinetics. MAS, mannitol‐sucrose buffer; DLD, D‐lactate:cytochrome c oxidoreductase; TCA, tricarboxylic acid cycle; Q, coenzyme Q; Cytc, cytochrome c; II, succinate dehydrogenase; III, coenzyme Q:cytochrome c‐oxidoreductase; IV, cytochrome c oxidase.