Abstract
The lateral segregation of membrane constituents into functional microdomains, conceptually known as lipid raft, is a universal organization principle for cellular membranes in both prokaryotes and eukaryotes. The widespread Stomatin, Prohibitin, Flotillin, and HflK/C (SPFH) family proteins are enriched in functional membrane microdomains at various subcellular locations, and therefore were hypothesized to play a scaffolding role in microdomain formation. In addition, many SPFH proteins are also implicated in highly specific processes occurring on the membrane. However, none of these functions is understood at the molecular level. Here we report the structure of a supramolecular complex that is isolated from bacterial membrane microdomains and contains two SPFH proteins (HflK and HflC) and a membrane-anchored AAA+ protease FtsH. HflK and HflC form a circular 24-mer assembly, featuring a laterally segregated membrane microdomain (20 nm in diameter) bordered by transmembrane domains of HflK/C and a completely sealed periplasmic vault. Four FtsH hexamers are embedded inside this microdomain through interactions with the inner surface of the vault. These observations provide a mechanistic explanation for the role of HflK/C and their mitochondrial homologs prohibitins in regulating membrane-bound AAA+ proteases, and suggest a general model for the organization and functionalization of membrane microdomains by SPFH proteins.
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Data availability
The cryo-EM map and atomic model of the HflK–HflC–FtsH complex have been deposited in the EMDB and PDB with accession codes EMDB-32002 and 7VHP, respectively. The map and model of a quarter of the complex used for symmetry expansion have been deposited in the EMDB and PDB with accession codes EMDB-32003 and 7VHQ, respectively. All data needed to support the conclusions in this study are included in the main text and supplementary materials.
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Acknowledgements
We thank the Core Facilities at the School of Life Sciences, Peking University for help with negative staining EM; the Cryo-EM Platform and the Electron Microscopy Laboratory of Peking University for help with data collection; the High-performance Computing Platform of Peking University for help with computation; and the National Center for Protein Sciences at Peking University for assistance in key experiments. The work was supported by the National Natural Science Foundation of China (31725007 and 31630087 to N.G.; 31922036 to N.L.; 31800625 to C.M.), the National Key R&D Program of China (2019YFA0508904 to N.G.), the Qidong-SLS Innovation Fund (to N.G.), and the China Postdoctoral Science Foundation (2018M631249 and 2021T140016 to C.M.). C.M. and C.W. were supported in part by the Postdoctoral Fellowship of Peking-Tsinghua Center for Life Sciences.
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N.G. designed and supervised the project; C.W. purified the sample with help from D.L., L.Y. and W.Y. and performed protease and pull-down assays; C.M. processed cryo-EM data with help from N.L.; N.G. built atomic models and analyzed the structure with C.M. and C.W.; C.M. prepared figures; N.G. wrote the manuscript; C.W. and C.M. revised the manuscript. All the authors approved the manuscript.
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Ma, C., Wang, C., Luo, D. et al. Structural insights into the membrane microdomain organization by SPFH family proteins. Cell Res 32, 176–189 (2022). https://doi.org/10.1038/s41422-021-00598-3
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DOI: https://doi.org/10.1038/s41422-021-00598-3
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