Abstract
The P4 ATPases use ATP hydrolysis to transport large lipid substrates across lipid bilayers. The structures of the endosome- and Golgi-localized phosphatidylserine flippases—such as the yeast Drs2 and human ATP8A1—have recently been reported. However, a substrate binding site on the cytosolic side has not been found, and the transport mechanisms of P4 ATPases with other substrates are unknown. Here we report structures of the S. cerevisiae Dnf1–Lem3 and Dnf2–Lem3 complexes. We captured substrate phosphatidylcholine molecules on both the exoplasmic and cytosolic sides and found that they have similar structures. Unexpectedly, Lem3 contributes to substrate binding. The conformational transitions of these phosphatidylcholine transporters match those of the phosphatidylserine transporters, suggesting a conserved mechanism among P4 ATPases. Dnf1/Dnf2 have a unique P domain helix-turn-helix insertion that is important for function. Therefore, P4 ATPases may have retained an overall transport mechanism while evolving distinct features for different lipid substrates.
Data availability
The cryo-EM 3D maps and the corresponding atomic models of the Dnf1-Lem3 complex have been deposited at the EMDB database and the RCSB PDB with the respective accession codes of EMD-23069 and 7KY6 (apo E1), EMD-23074 and 7KYB (E1P-ADP), EMD-23077 and 7KYC (E2P). The cryo-EM 3D maps and the corresponding atomic models of the Dnf2-Lem3 complex have been deposited at the EMDB database and the RCSB PDB with the respective accession codes of EMD-23070 and 7KY7 (apo E1), EMD-23071 and 7KY8 (E1-ATP), EMD-23072 and 7KY9 (E1P-ADP), EMD-23068 and 7KY5 (E2P-transition), and EMD-23073 and 7KYA (E2P).
Article and author information
Author details
Funding
National Institutes of Health (CA231466)
- Huilin Li
National Institutes of Health (GM107978)
- Todd R Graham
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Copyright
© 2020, Bai et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
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Transport mechanism of P4 ATPase phosphatidylcholine flippases
eLife 9:e62163.
https://doi.org/10.7554/eLife.62163
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Liquid-liquid phase separation (LLPS) involving intrinsically disordered protein regions (IDRs) is a major physical mechanism for biological membraneless compartmentalization. The multifaceted electrostatic effects in these biomolecular condensates are exemplified here by experimental and theoretical investigations of the different salt- and ATP-dependent LLPSs of an IDR of messenger RNA-regulating protein Caprin1 and its phosphorylated variant pY-Caprin1, exhibiting, for example, reentrant behaviors in some instances but not others. Experimental data are rationalized by physical modeling using analytical theory, molecular dynamics, and polymer field-theoretic simulations, indicating that interchain ion bridges enhance LLPS of polyelectrolytes such as Caprin1 and the high valency of ATP-magnesium is a significant factor for its colocalization with the condensed phases, as similar trends are observed for other IDRs. The electrostatic nature of these features complements ATP’s involvement in π-related interactions and as an amphiphilic hydrotrope, underscoring a general role of biomolecular condensates in modulating ion concentrations and its functional ramifications.
