Luis Daniel Cruz‐Zaragoza

Mitochondria play central roles in cellular energy production and metabolism. Most proteins required to carry out these functions are synthesized in the cytosol and imported into mitochondria. A growing number of metabolic disorders arising from mitochondrial dysfunction can be traced to errors in mitochondrial protein import. The mechanisms underlying the import of precursor proteins are commonly studied using radioactively labeled precursor proteins imported into purified mitochondria. Here, we establish a fluorescence‐based import assay to analyze protein import into mitochondria. We show that fluorescently labeled precursors enable import analysis with similar sensitivity to those using radioactive precursors, yet they provide the advantage of quantifying import with picomole resolution. We adapted the import assay to a 96‐well plate format allowing for fast analysis in a screening‐compatible format. Moreover, we show that fluorescently labeled precursors can be used to monitor the assembly of the F 1 F 0 ATP synthase in purified mitochondria. Thus, we provide a sensitive fluorescence‐based import assay that enables quantitative and fast import analysis.