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.
Highlights
The large group of integral membrane proteins that mediate the transport of substrates across biological membranes by the formation of ATP-dependent phosphorylated intermediates are called P-type ATPases (Axelsen and Palmgren, 1998)
Most P4 ATPases function as a heterodimeric complex: a catalytic a-subunit containing 10 transmembrane a-helices (TMHs) and a regulatory b-subunit of the Cdc50 protein family containing two TMHs
Chief among them are how substrate specificity is achieved by different P4 ATPases and where the lipid binds on the cytoplasmic side of the membrane
Summary
The large group of integral membrane proteins that mediate the transport of substrates across biological membranes by the formation of ATP-dependent phosphorylated intermediates are called P-type ATPases (Axelsen and Palmgren, 1998). P4 ATPases are involved in phospholipid translocation: they flip lipids from the extracellular side of the plasma membrane or from the lumenal side of internal organelles to the cytosolic side (Tang et al, 1996; Zhou and Graham, 2009). Chief among them are how substrate specificity is achieved by different P4 ATPases and where the lipid binds on the cytoplasmic side of the membrane Addressing these questions requires a detailed structural and functional comparison among P4 ATPases that transport different substrates. Our comprehensive structural and functional study improves the mechanistic understanding of the essential P4 ATPases
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