Yan Youmian, Erdenepurev Baigalmaa, Collinson Ian, Niemi Natalie M
Department of Biochemistry & Molecular Biophysics, Washington University School of Medicine, St. Louis, MO 63110, USA.
School of Biochemistry, University of Bristol, Bristol BS8 1TD, United Kingdom.
bioRxiv. 2025 May 29:2025.05.26.655807. doi: 10.1101/2025.05.26.655807.
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), 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.
数百种定位于线粒体的蛋白质依靠N端前导序列进行细胞器靶向和导入。虽然前导序列通常被描述为带正电荷的两亲性螺旋，但它们缺乏一致的基序，因此可能以不同的效率促进蛋白质导入线粒体。事实上，前导序列“强度”的概念是诸如应激感应等生物学模型的关键基础，但目前缺乏对决定“强”前导序列与“弱”前导序列因素的定量分析。此外，前导序列强度对线粒体功能和细胞适应性的影响程度仍不清楚。在这里，我们利用MitoLuc线粒体蛋白质导入测定的高通量和动力学特性来量化前导序列强度的多个方面。我们发现，选定的前导序列，包括那些调节线粒体未折叠蛋白反应（UPR）的序列，足以在 mitochondrial uncoupling 期间赋予不同的导入效率。令人惊讶的是，我们发现在体外应激和基础（即非应激）条件下，除了那些经典上与应激信号相关的前导序列之外，其他前导序列会促进高度可变的导入效率，这表明前导序列强度可能影响比目前所认识到的更广泛的一系列过程。我们利用这种变异性来证明，只有在体外促进强大导入的前导序列才能完全挽救复合物IV缺陷酵母呼吸生长中的缺陷，这表明前导序列强度决定代谢潜力。总的来说，我们的研究结果表明，前导序列强度可以描述许多指标，如总导入蛋白、最大导入速度或对解偶联的敏感性，这表明将前导序列注释为“弱”与“强”需要比通常更细致的表征。重要的是，我们发现在应激信号之外的过程中，前导序列强度的这种变异性对细胞适应性有显著影响，这表明生物体可能广泛利用前导序列强度来微调线粒体导入，从而调节细胞器稳态。
