Yeast-Based High-Throughput Screening Platform for Identifying Modulators of Mitochondrial Protein Import

Mitochondrial protein import is essential for overall cellular homeostasis, yet scalable approaches to systematically interrogate mitochondrial protein import and identify modulators of this process remain limited. Here, we describe a yeast-based, gain-of-growth (GoG), high-throughput screening assay for the identification of small-molecule modulators of mitochondrial protein import. In this system, truncated human proteins that contain N-terminal mitochondrial targeting sequences (MTSs) are expressed in S. cerevisiae, where mitochondrial protein import is coupled to an auxotrophic growth readout. Disruption of import leads to cytosolic accessibility of the URA3 reporter, producing a GoG phenotype under selective conditions. As a proof of concept, we applied this model to PTEN-induced kinase 1 (PINK1), a mitochondrial imported regulator of mitochondrial quality control. Using this approach, we demonstrate the ability to monitor PINK1 import and identify candidate compounds that modulate this process. Collectively, this work establishes a scalable and reproducible platform for interrogating mitochondrial protein import and identifying compounds for downstream validation in mammalian systems.