ORCID
https://orcid.org/0000-0001-8733-6989
Language
English (en)
Date of Award
12-15-2026
Degree Type
Dissertation
Degree Name
Doctor of Philosophy (PhD)
Chair and Committee
Natalie M. Niemi
Committee Members
Eric A. Galburt, Alex S. Holehouse, Shankar Mukherji, David J. Pagliarini
Abstract
Mitochondria are essential organelles housing over 1000 proteins, 99% of which are nuclear-encoded and synthesized in the cytosol. Most mitochondrial proteins are targeted by a positively charged sequence called the presequence, which lacks a common sequence motif and is poorly conserved across evolution. Accumulating evidence suggests that presequences encode meaningful information that influences mitochondrial protein uptake. For instance, multiple stress response models propose that stress sensing proteins contain “weak” presequences which allows them to monitor mitochondrial health. However, there is a lack of systematic examination about how presequences influence import kinetics and what constitutes presequence strength. These gaps in knowledge partially derive from methodological limitations, as mitochondrial import is typically measured by low-throughput methods with poor kinetic resolution.
By optimizing a newly developed in vitro import assay based on split-luciferase complementation, the MitoLuc assay, we have established a framework to measure mitochondrial protein import kinetics through parameters such as amount of protein imported, rate of protein import, lag time, and sensitivity towards loss of membrane potential, thus providing a quantitative dissection of presequence strength. We also combined the MitoLuc assay with a potentiometric dye to enable simultaneous monitor of mitochondrial protein import and membrane potential for the first time. Using this suite of tools, we have showed that varied presequences are sufficient to promote a wide range of mitochondrial import efficiencies when attached to a common cargo protein under both basal conditions and uncoupling conditions. Importantly, only presequences promoting efficient in vitro import could fully rescue the defects in respiratory growth of a strain of complex IV-deficient yeast, emphasizing the differences in presequence strength meaningfully affect cellular fitness.
The quantitative nature of the MitoLuc assay allows us to finely compare mitochondrial protein import kinetics in multiple aspects and identify differences that emerge under selective conditions. To better understand the mechanisms by which presequences impart differential import efficiency and the molecular processes quantified by the MitoLuc assay, we perturbed import through chemical denaturation of the precursor proteins and compared the changes in import kinetics. We found that precursor unfolding is the rate-limiting step in the MitoLuc assay, and this unfolding rate is affected by presequences in a way that correlates with their amphiphilicity. The amphiphilicity of presequences mainly exerts its effect by driving the translocation across the inner membrane of mitochondria down the charge gradient. In addition, presequences still caused varied import efficiency of denatured precursor proteins, potentially by affecting the initial association with mitochondria.
Lastly, using the toolkit we developed, we characterized the targeting mechanism of the mitochondrial phosphatase PPTC7, whose dual localization to the outer mitochondrial membrane and matrix has been attributed to its weak presequence. Surprisingly, we found that the presequence of PPTC7 is relatively weak but insufficient to promote dual localization. In contrast to other known weak presequences, PPTC7 presequence is not particularly sensitive to change in mitochondrial membrane potential. A disordered glycine-rich region downstream of the presequence negatively affects the matrix import capacity potentially by decreasing the helical potential of the PPTC7 presequence. In addition, the phosphatase domain of PPTC7 is highly stable and challenging to import, where chemical denaturation coupled with a strong presequence is needed for in vitro import. These results not only underscore the multifactorial sequence elements that control the localization of PPTC7 but also emphasize that the functionality of presequences can be fine-tuned by downstream sequence contexts.
Collectively, our works indicate that presequences are sufficient to give rise to differential protein import behavior under both basal and stressed conditions, and the wide variations across multiple kinetic parameters further emphasize the importance of a precise definition of presequence strength. Amphiphilicity has the highest predictive power for presequence strength based on primary sequences, potentially through driving mitochondrial association and precursor unfolding by the membrane potential. Importantly, the targeting capacity of presequences could be altered by downstream sequences, thus their native sequence contexts should be considered individually for physiological applications.
DOI
https://doi.org/10.48765/pgfd-rq56
Recommended Citation
Yan, Youmian, "Investigating the Ability of Presequences to Influence Mitochondrial Protein Import" (2026). WUSM Theses and Dissertations – All Programs. 61.
https://digitalcommons.wustl.edu/all_etd/61
