Abstract
Phage shock protein A (PspA), a recently identified member of the endosomal sorting complex required for transport III (ESCRT-III) superfamily, is proposed to be critical in stress-induced membrane remodeling in bacteria; however, the precise function and mechanism remain largely unexplored. In this study, we employed various lipid membrane models, including giant unilamellar vesicles, small unilamellar vesicles, and supported lipid bilayers, to study membrane-related activities of PspA. Through cell-free protein synthesis of PspA and biophysical characterizations, we demonstrated its capacity to self-assemble and uncovered the decisive role of the α1 region in facilitating this self-assembly. Notably, assays using lipid bilayer-based systems revealed a range of membrane-associated activities, including binding, disruption, and remodeling, encompassing membrane tubulation, elongation, and the formation of double-membrane vesicles. We discovered that the K55 residue within the α1 region is a key determinant for lipid binding and observed its effects on overall membrane remodeling activities, while the R44 residue is crucial for forming stable PspA rods. Together, these findings highlight the importance of the α1 domain in mediating membrane remodeling activity and suggest this may serve as a viable mechanism for other ESCRT-III proteins.