bioRxiv. 2025 May 29. pii: 2025.05.26.655807. [Epub ahead of print]
Hundreds of mitochondrial-destined proteins rely on N-terminal presequences for organellar targeting and import. While generally described as positively charged amphipathic helices, presequences lack a consensus motif and thus likely promote the import of proteins into mitochondria with variable efficiencies. Indeed, the concept of presequence "strength" critically 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 high-throughput and kinetic nature of the MitoLuc mitochondrial protein import assay to quantify multiple aspects of presequence strength. We find that select presequences, including those that regulate the mitochondrial unfolded protein response (UPR mt ), are sufficient to impart differential import efficiencies during mitochondrial uncoupling. Surprisingly, we find that presequences beyond those classically associated with stress signaling promote highly variable import efficiency in stressed and basal (i.e., non-stressed) conditions in vitro, suggesting that presequence strength may influence a broader array of processes than currently appreciated. We exploit this variability to demonstrate that only presequences that promote robust import in vitro 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" versus "strong" requires more nuanced characterization than is typically performed. Importantly, we find that such variability in presequence strength meaningfully affects cellular fitness in processes beyond stress signaling, suggesting that organisms may broadly exploit presequence strength to fine-tune mitochondrial import and thus organellar homeostasis.