Investigating Mitochondrial Presequence Import
by Naintara Jain
Date of Examination:2025-08-06
Date of issue:2025-10-29
Advisor:Prof. Dr. Peter Rehling
Referee:Prof. Dr. Peter Rehling
Referee:Dr. Alex Caspar Faesen
Persistent Address:
http://dx.doi.org/10.53846/goediss-11596
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Name:Naintara Jain_PhD Dissertation.pdf
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Description:PhD Dissertation submitted by Naintara Jain (2025)
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Abstract
English
Mitochondrial biogenesis and function depend on the accurate import of nuclear-encoded proteins via specialized translocase complexes. Disruptions in these import pathways are implicated in numerous mitochondrial disorders, particularly in energy-demanding tissues. Around 70% of mitochondrial proteins are delivered through the coordinated action of the TOM complex in the outer membrane and the TIM23 complex in the inner membrane. This thesis investigates protein import via the TOM–TIM23 pathway, aiming to develop experimental tools that enable mechanistic and structural insights under near-native conditions. The first part of this work presents a fluorescence-based in organello import assay adapted from classical radiolabeled methods. Using DyLight-labeled model precursors such as Jac1 and Atp5, the assay enables quantitative analysis of import kinetics without the need for radioactivity. The method allows absolute quantification of imported protein in picomolar ranges and supports rapid, high-throughput workflows via SDS–PAGE or 96-well plate-based fluorescence detection. In addition to faithfully replicating the sensitivity and resolution of traditional assays, it permits direct comparison of purified precursors to probe import machinery dynamics, and has proven effective in studying presequence properties and downstream assembly steps. In the second part of the thesis, a strategy to biochemically isolate the TOM–TIM23 supercomplex in a substrate-engaged state was optimized and refined. A model precursor (Jac1–sfGFP), designed to arrest during import, was used to trap the complex. Various affinity tag combinations were tested, and a dual-tag system (Tim23–SUMO*–His and Jac1–sfGFP–FLAG) combined with chemical crosslinking was optimized to enrich for stable translocation intermediates. Although the resulting samples showed improved biochemical stability, cryo-EM analysis was hindered by particle heterogeneity and low contrast. To reduce configurational variability and improve retention of matrix-facing components, a Tim17–Pam18 fusion construct was employed to stabilize the TIM23MOTOR conformation. Additionally, in vivo expression of the clogging precursor was explored as a less disruptive alternative to in vitro import, reducing sample handling while preserving physiological relevance. Combining these approaches—genetic fusion, in vivo stalling, immunoisolation, and mild crosslinking—could lead to an increase in the enrichment of stalled TOM-TIM23 supercomplexes and forms the basis for a future pipeline geared toward cryo-EM analysis. Together, this work introduces a robust, quantitative import assay, and an improved biochemical strategy for capturing substrate-engaged mitochondrial translocase complexes. These tools can complement existing structural and biochemical approaches in the field, and support future efforts to dissect the dynamic, compositionally flexible nature of the TOM–TIM23 import machinery under controlled experimental conditions.
Keywords: Mitochondria; Presequence pathway; Fluorescent Import Assay; TOM-TIM23 Supercomplex; Cryo-EM
