F1F0-ATP-synthase subunit b head-to-head interactions shape intracellular membranes in Escherichia coli

Intracellular membrane (ICM) compartments in bacteria remain poorly understood. In Escherichia coli, ICMs form upon the overproduction of certain membrane proteins. Here, we investigate at the molecular level how overproduction of subunit b of the F1Fo-ATP synthase complex shapes ICMs. Through a combination of subunit b deletions, electron microscopy, and biochemical analyses, we demonstrate that ICM network structuration is mediated by the C-terminal domain of subunit b. High-resolution cryo-electron tomography and molecular dynamics simulations reveal that this domain forms tetrameric assemblies, bridging membranes and maintaining a regular 26 nm spacing. Transcriptomic and proteomic analyses further show that the cell responds to ICM formation by upregulating energy metabolism genes and the ESCRT-III homolog PspA, while downregulating ribosomal and translational machinery. These results uncover a novel mechanism by which the C-terminal domain of subunit b drives the structuring of intracellular membranes (ICMs). Additionally, they highlight how E. coli adapts to membrane stress by overexpressing membrane-remodeling proteins, a response that shares similarities with intracellular membrane homeostasis mechanisms in photosynthetic bacteria and unicellular algae.