Transport mechanism of P4 ATPase phosphatidylcholine flippases

Acceptance summary:

Your study nicely shows that PC and PS flippases use a common mechanism for flipping these lipids between leaflets of biological membranes.

Decision letter after peer review:

Thank you for submitting your article "Transport mechanism of class-3 P4 ATPase lipid flippases" for consideration by eLife. Your article has been reviewed by three peer reviewers, including Christopher G Burd as the Reviewing Editor and Reviewer #1, and the evaluation has been overseen by Vivek Malhotra as the Senior Editor.

The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.

As the editors have judged that your manuscript is of interest, but as described below that additional experiments are required before it is published, we would like to draw your attention to changes in our revision policy that we have made in response to COVID-19 (https://elifesciences.org/articles/57162). First, because many researchers have temporarily lost access to the labs, we will give authors as much time as they need to submit revised manuscripts. We are also offering, if you choose, to post the manuscript to bioRxiv (if it is not already there) along with this decision letter and a formal designation that the manuscript is "in revision at eLife". Please let us know if you would like to pursue this option. (If your work is more suitable for medRxiv, you will need to post the preprint yourself, as the mechanisms for us to do so are still in development.)

Summary:

The manuscript presents a structural analysis of the transport cycle of P4 ATPases, which mediate phospholipid translocation between leaflets of biological membranes. Eight structures of two yeast class-3 P4 ATPases, Dnf1- Lem3 and Dnf2-Lem3, in five transport states were determined using cryo-electron microscopy methods. Structure-based mutagenesis of mutant enzymes expressed in yeast cells were used to test and validate mechanistic predictions based on the structures. While the overall structures or Dnf1- Lem3 and Dnf2-Lem3 are quite similar to previously determined structures of these and other P4 ATPases, several new pieces of information were obtained. Most important is the identification of two lipid binding sites that are inferred to define the sites of substrate entry on the exofacial membrane leaflet and substrate exit on the cytoplasmic leaflet. A second major point is the inference of an unexpected role for the Lem3 β subunit in substrate recognition and inter-domain contacts that regulate transport activity. Finally, the identification of the conformational transitions during substrate flipping revealed that they are essentially identical to that of the class^-1 P4 ATPases, therefore indicating that the pathways and mechanisms involved in lipid flipping are likely identical between enzymes in all classes.

Essential revisions:

1) Recent phylogenetic analysis revealed a new classification that groups Dnf1/2, ATP8A1 and Drs2 into the same class (Palmgren et al., 2019). This study should be cited in the manuscript and the revised classification considered in the interpretation of the results.

2) It is essential that the study provide evidence that the purified proteins under investigation display ATPase activity that can be stimulated by substrate lipids (but not by non-substrate lipids) and blocked by the ATP analogs/inhibitors. Published structural studies of relevant flippases (Bai et al., 2019, Timcenko et al., 2019, Hiraizumi et al., 2019) each include a demonstration the proteins under investigation are active and the reviewers consider this a standard that should be maintained. At a minimum, the authors should measure ATPase activity of the native sequence proteins in the presence/absence of ATP analogs/inhibitors.

3) The functional analyses of flippase mutants correlate changes in lipid uptake activity with specific mutations and attribute differences in lipid uptake activity to these mutations. The authors should establish if differences in expression levels of the mutant proteins can account (or not) for the observed changes in lipid uptake activity. A standard approach is to compare protein levels in cell extracts by immune-blotting. Such an analysis (or equivalent) should be included, if not for all the proteins tested, at least the ones that are most relevant to the authors conclusions.