Aberrant Topologies of Bacterial Membrane Proteins Revealed by High Sensitivity Fluorescence Labelling

The cytoplasmic membrane compartmentalises the bacterial cell into cytoplasm and periplasm. Proteins located in this membrane have a defined topology that is established during their biogenesis. However, the accuracy of this fundamental biosynthetic process is unknown. We developed compartment -specific fluorescence labelling methods with up to single-molecule sensitivity. Application of these meth-ods to the single and multi-spanning membrane proteins of the Tat protein transport system revealed rare topogenesis errors. This methodology also detected low level soluble protein mislocalization from the cytoplasm to the periplasm. This study shows that it is possible to uncover rare errors in protein localiza-tion by leveraging the high sensitivity of fluorescence methods.(c) 2023 The Author(s). Published by Elsevier Ltd.This is an open access article under the CC BY license (http://creativecom-mons.org/licenses/by/4.0/).