ISBN 9783868538885

978-3-86853-888-5, Reihe Biologie

Mercedes Romero-Ruiz
Interactions of Polypeptides with the Protein Translocation Channel of the Outer Membrane of Mitochondria

164 Seiten, Dissertation Universität Stuttgart (2010), Softcover, A5

Zusammenfassung / Abstract

The mitochondrial outer membrane protein translocase, the so-called TOM complex, plays an important role in the mitochondrial import machinery. Quantitative analysis about the import kinetics and the mechanism of substrate interaction with TOM was hampered by the unavailability of suitable protein import systems which allow monitoring substrate-TOM interactions at a single molecule level and with sufficiently high temporal resolution.

The aim of this study was to shed light onto the polypeptide translocation mechanism of the TOM machinery at a molecular level, providing a first quantitative analysis of its kinetics. In this study, I have subjected the TOM machinery of the filamentous fungus "Neurospora crassa" to high temporal resolution electrophysiological measurements.

TOM is a highly dynamic channel that can switch between a complex set of distinct conformational states. The largest conductance state of the translocon represents the likely protein-conducting conformation of the complex.

The first strategy to study the translocation mechanism of TOM-complex was to observe substrate-induced blockage of the main open conductance state. Closures of the channel were observed in the presence of the mitochondrial presequence pF1β and the synthetic model peptide pAK5. pF1β caused a total blockage of TOM at high voltages. To obtain kinetic data for peptide-TOM interaction, further experiments were performed in the presence of the synthetic model peptide pAK5. Channel blockage was concentration- and voltage-dependent which was superimposed on the endogenous channel gating. The data could be explained in terms of a modified Woodhull blocking mechanism.

The second strategy to monitor substrate-TOM interactions and gain information on the kinetics displayed by them was to perform single channel measurements at low voltages where the channel shows only little endogenous gating. New voltage-protocols and a new electrophysiological chamber with minimized membrane capacitance and noise signals were used. At constant low voltages, channel blockage was observed in the presence of pF1β at a single-molecule level. The frequency of channel blockage progressively increased with peptide concentration and was dependent on the membrane voltage indicating an open blocker mechanism. My data provided the rate constants of substrate association and dissociation and first glimpse into the kinetics of protein translocation through the mitochondrial TOM machinery.