The bacterial holo-translocon and friends

Abstract

All organisms are compartmentalised by membranes; in Gram-negative bacteria two membranes surround the cell with the peptidoglycan layer between them, together this forms the cell envelope. Protein translocation across the inner membrane for secretion, or insertion into the inner membrane is primarily conducted using the highly conserved, hourglass-shaped channel, SecYEG. It is now well established that to facilitate translocation SecYEG interacts with ancillary subcomplexes of SecDF-YajC and YidC to form a complex termed the holo-translocon (HTL). Despite this, the majority of previous publications have focused primarily on the core SecY channel in complex with interacting cytoplasmic proteins. This thesis takes a structural approach toward establishing the network of different protein interactions within the cytoplasm, bacterial inner membrane, periplasm and outer membrane that work cooperatively to orchestrate protein translocation of a broad range of secretory substrates. We conducted electron cryo-microscopy (cryo-EM) of the holo-translocon in detergent-free membrane mimetic nanodiscs to investigate the structure of the translocon in a native lipid environment. Density gradient ultra- centrifugation and fixation techniques were used for isolation of these multi-protein complexes. Negative-stain electron microscopy to establish and characterise interactions between the holo-translocon, periplasmic chaperone SurA and the cytoplasmic ATPase SecA. Finally electron cryo-microscopy revealed a low resolution structure of the substrate-engaged SurA bound to the holo-translocon. This thesis establishes a role for holo-translocon in SecA-dependent translocation through the inner membrane and expands on the SecY-centric view of protein translocation to investigate the dynamic interactions that aid secretion.

Date of Award	28 Sept 2021
Original language	English
Awarding Institution	University of Bristol
Supervisor	Ian R Collinson (Supervisor) & Imre Berger (Supervisor)