The structure and function of P5A-ATPases

In eukaryotes P5A-ATPases are resident in the endoplasmic reticulum (ER) membrane. Their dysfunction causes broad and unspecific phenotypes, with effects on protein biogenesis and quality control. While P5A-ATPases have been suggested to remove misinserted proteins from the membrane, the molecular function remains debated. Here, we report cryo-EM structures reaching 3.2 Å overall resolution of a eukaryotic P5A-ATPase member, CtSpf1, covering multiple transport intermediates of the E1→E1-ATP→E1P-ADP→E1P→E2P→E2.Pi→E2 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 into the central P-domain, here referred to as the Plug-domain. The Plug-domain is nestled to key ATPase features and is displaced in the E1P-ADP and E1P states. Interestingly, a complementary cryo-EM feature is detected also in the E1P-ADP conformation, located peripherally to the above-mentioned cargo. Collectively, our structural and mass spectrometry data are compatible with a broad range of proteins as cargo, with the P5A-ATPases serving a role in membrane removal, although membrane insertion/secretion cannot be excluded. Our work also reveals a critical mechanistic role of the Plug-domain and certain N-terminal transmembrane segments, where the latter couple conformational changes to structural changes of the soluble domains.