Single Molecule Measurements on the Mechanism of Protein Import by the Toc Translocon

Abstract

In cells up to 50% of all cytosolic synthesized proteins have to traverse at least one membrane to reach their place of function. This requires the action of membrane embedded molecular machines. The Toc complex in chloroplasts is such a molecular machine (Soll, J. and Schleiff, E. Nat Rev Mol Cell Biol, 2004). Despite the identification of many components involved in the translocation process, not much is known about the mode of function of the Toc complex at the molecular level. We address this molecular mechanism with Single Molecule Fluorescence Resonance Energy Transfer (smFRET) and magnetic tweezers (MT) experiments. Specifically labelled components of the Toc complex and fluorescent labelled GTP (analogues) give insights into the stoichiometry and assembly of the Toc complex as well as its GTPase function under various conditions. The stall forces, force generating steps and translocation velocities are deduced from MT experiments. Finally, a combination of these two methods will allow to formulate a kinetic model of protein translocation, which will be an important step for the biological and systematic description of protein sorting and, subsequently, cellular function.