Hundreds of mitochondrial proteins rely on N-terminal presequences for organellar targeting and import. While generally described as positively charged amphiphilic helices, presequences lack a consensus motif and thus likely promote protein import into mitochondria with variable efficiencies. Indeed, the concept of presequence strength underlies biological models such as stress sensing, yet a quantitative analysis of what dictates strong versus weak presequences is lacking. Furthermore, the extent to which presequence strength affects mitochondrial function and cellular fitness remains unclear. Here, we capitalize on the MitoLuc protein import assay to define multiple aspects of presequence strength. We find that select presequences, including those that regulate the mitochondrial unfolded protein response (UPRmt), impart differential import efficiencies during mitochondrial uncoupling. Surprisingly, we find that presequences beyond those associated with stress signaling promote highly variable import efficiency in vitro, suggesting presequence strength may influence a broader array of processes than currently appreciated. We exploit this variability to demonstrate that only presequences that promote robust in vitro import can fully rescue defects in respiratory growth in complex IV–deficient yeast, suggesting that presequence strength dictates metabolic potential. Collectively, our findings demonstrate that presequence strength can describe numerous metrics, such as total imported protein, maximal import velocity, or sensitivity to uncoupling, suggesting that the annotation of presequences as weak or strong requires more nuanced characterization than typically performed. Importantly, we find that such variability in presequence strength meaningfully affects cellular fitness beyond stress signaling, suggesting that organisms may broadly exploit presequence strength to fine-tune mitochondrial import and thus organellar homeostasis.
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