Molecular Characterization of the Mitochondrial Presequence Translocase

by Niels Denkert
Doctoral thesis
Date of Examination:2017-11-24
Date of issue:2018-01-31
Advisor:Prof. Dr. Michael Meinecke
Referee:Prof. Dr. Claudia Steinem
Referee:Prof. Dr. Ralph Kehlenbach
Referee:Prof. Dr. Peter Rehling
Referee:Prof. Dr. Stefan Jakobs
Referee:Dr. Alexander Stein
Persistent Address: http://dx.doi.org/10.53846/goediss-6710

 

 

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Abstract

English

In this thesis, multiple pore-lining amino acid residues of the mitochondrial presequence translocation channel Tim23 were identified as constituents of the channel’s cation filter by combining single channel electrophysiology and site-directed mutagenesis. Unlike proposed before, the ion filter cannot be constituted by a localized constriction zone within the channel, but possibly by providing an energetically favorable or unfavorable surface pathway for ions, spanning the whole channel lumen. Using electrophysiology and yeast biochemistry, we showed that the cation preference is a key property in recognizing and especially translocating positively charged presequences in vitro and preproteins in organello. High-resolution analysis of electrophysiology data further indicates that the presequence-induced fast-gating state of Tim23 presumably corresponds to a translocating state with peptides in transit. Further, we investigated the domain origin for critical functions of the main receptor Tim50. We could show that both voltage regulation of, and presequence handover to Tim23 is independent of the essential presequence binding domain PBD but is localized in the soluble core domain of Tim50.
Keywords: Tim23; electrophysiology; protein translocation; ion selectivity
 
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