Single molecule studies on protein translocation in Escherichia coli

Proteins are the working horses of the cell. They are responsible for many events such as building, regulation, locomotion and signaling. Proteins are mainly synthesized in the cytoplasm. Frequently, during or after the synthesis, the protein needs to be transported across a lipid membrane to the different cell compartments or outside of the cell. In the bacterial kingdom the main translocation pathway is provided by the Sec translcocase. In Escherichia coli the translocase consists of a protein conducting channel (PCC) formed by the SecY, SecE and SecG proteins, and several associated proteins, among which the motor protein SecA, an ATPase. In post translational protein translocation, the preprotein associates with the cytosolic chaperone SecB, during or just after its synthesis. SecB is a secretion-dedicated chaperone that fulfills two functions: to maintain preproteins in a translocation competent state and to target preproteins to the SecYEG bound SecA at the cis-side of the membrane. Upon a successful interaction between the preprotein and SecA, multiple cycles of ATP binding and hydrolysis by SecA result in the progressive movement of preprotein segments through the PCC. The proton motive force (PMF) provides an additional energy source for translocation The goal of the work described in this thesis was to investigate the mechanism of protein translocation by the preprotein translocase of Escherichia coli, with the ultimate aim to analyze this process at the single molecule level.