Abstract
Endoplasmic reticulum (ER) membrane resident P5A-ATPases broadly affect protein biogenesis and quality control, and yet their molecular function remains debated. Here, we report cryo-EM structures of a P5A-ATPase, CtSpf1, covering multiple transport intermediates of the E1 → E1-ATP → E1P-ADP → E1P → E2P → E2.Pi → E2 → E1 cycle. In the E2P and E2.Pi states a cleft spans the entire membrane, holding a polypeptide cargo molecule. The cargo includes an ER luminal extension, pinpointed as the C-terminus in the E2.Pi state, which reenters the membrane in E2P. The E1 structure harbors a cytosol-facing cavity that is blocked by an insertion we refer to as the Plug-domain. The Plug-domain is nestled to key ATPase features and is displaced in the E1P-ADP and E1P states. Collectively, our findings are compatible with a broad range of proteins as cargo, with the P5A-ATPases serving a role in membrane removal of helices, although insertion/secretion cannot be excluded, as well as with a mechanistic role of the Plug-domain.
| Originalsprog | Engelsk |
|---|---|
| Artikelnummer | 9605 |
| Tidsskrift | Nature Communications |
| Vol/bind | 15 |
| Udgave nummer | 1 |
| Antal sider | 15 |
| ISSN | 2041-1723 |
| DOI | |
| Status | Udgivet - 2024 |
Bibliografisk note
Funding Information:This study was supported by the Swedish Research Council (2016-04474 and 2022-01315; P.G.), Knut and Alice Wallenberg Foundation (2015.0131 and 2020.0194; P.G.), The Lundbeck Foundation (R322-2019-2588; VB, R324-2019-1855; KW, R218-2016-1548, R133-A12689, R313-2019-774 and R346-2020-2019; PG), the Danish Council for Independent Research (9039-00273 and 3101-00303; P.G.), Carl Trygger Foundation (CTS 21:1773), the Crafoord Foundation (20170818, 20180652, 20200739 and 20220905; PG) and The Per-Eric and Ulla Schyberg Foundation (38267; P.G.). P.L. also obtained support from the Royal Physiographic Society of Lund. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the paper. We would like to thank Julian Conrad, Karin Wallden, Dustin Morado, and Marta Corrani at the Cryo-EM Swedish National Facility in Stockholm for sample screening and data collection. The Cryo-EM Swedish National Facility at SciLifeLab is funded by the Knut and Alice Wallenberg, Family Erling Persson and Kempe Foundations, SciLifeLab, Stockholm University, and Ume\u00E5 University. We also would like to thank Tillmann Hanns Pape at the Danish Cryo-EM Facility at the Core Facility for Integrated Microscopy (CFIM) at the University of Copenhagen for sample screening and data collection. The Danish Cryo-EM Facility at CFIM, University of Copenhagen, is supported by Novo-Nordisk Foundation grant id NNF14CC0001. We also acknowledge the Lund University Cryo-EM platform for access to the GPU computers. We would like to thank Profs. Per Amstrup Pedersen (University of Copenhagen) and Kefeng Lu (Sichuan University) for providing the PAP1500 and ScSpf1 knockout strains, respectively, and Kefeng Lu also for the cellular localization images. We also would like to thank Charlotte Welinder and Hong Yan at the Center for Translational Proteomics at the Medical Faculty, Lund University, for their assistance with the M.S.
Publisher Copyright:
© The Author(s) 2024.