Conserved mechanism of phospholipid substrate recognition by the P4-ATPase Neo1 from Saccharomyces cerevisiae

Huang, Yannan; Takar, Mehmet; Best, Jordan T.; Graham, Todd R.

doi:10.1016/j.bbalip.2019.158581

Cited by 9 publications

(6 citation statements)

References 54 publications

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“…Although the exact substrates a P4 ATPase transport differ, the substrate translocation path appears to have been conserved in all P4 ATPase studied so far (Andersen et al, 2016;Huang et al, 2020). Like the Drs2 and ATP8A1 flippases, we found that the Dnf1 substrate translocation path is composed of TMH1-4 and TMH6 ( Fig.…”

Section: Phospholipids Bind At the Exoplasmic And Cytoplasmic Sites Omentioning

confidence: 65%

Transport Mechanism of Class-3 P4 ATPase Lipid Flippases

Bai

You

Jain

et al. 2020

Preprint

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The P4 ATPases are a family of membrane enzymes that transport lipids across membrane bilayers. The structures of the class-1 phosphatidylserine flippases have been reported, revealing a substrate site on lumenal side. However, the cytosolic binding site has not been found, and the transport mechanisms of P4 ATPases in other classes are unknown. Here we report a structural and functional study on plasma-membrane localized, class-3 P4 ATPases Dnf1–Lem3 and Dnf2–Lem3. We captured substrate lipids on both the exoplasmic and cytosolic sides, and found that these two enzymes have similar structures. We found a helix-turn-helix motif in the cytosolic P domain that is unique to the class-3 enzymes and plays a crucial role in their function. Unexpectedly, Lem3 contributes to substrate binding near the cytosolic surface. Conformational transitions of these two class-3 enzymes match those of the class-1 enzymes, suggesting a conserved mechanism among all classes of P4 ATPases.

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Section: Phospholipids Bind At the Exoplasmic And Cytoplasmic Sites Omentioning

confidence: 65%

Transport Mechanism of Class-3 P4 ATPase Lipid Flippases

Bai

You

Jain

et al. 2020

Preprint

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“…S4 ). Other determinants of substrate specificity, flippase activity, and exit gate position in TM2 and TM4 ( 47 , 50 , 51 ) also differ among all shown P4-ATPases (boxed residues, Fig. S4 ).…”

Section: Resultsmentioning

confidence: 99%

Aminoglycerophospholipid flipping and P4-ATPases in Toxoplasma gondii

Chen

Günay-Esiyok

Klingeberg

et al. 2021

Journal of Biological Chemistry

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Lipid flipping in the membrane bilayers is a widespread eukaryotic phenomenon that is catalyzed by assorted P4-ATPases. Its occurrence, mechanism, and importance in apicomplexan parasites have remained elusive, however. Here we show that Toxoplasma gondii , an obligate intracellular parasite with high clinical relevance, can salvage phosphatidylserine (PtdSer) and phosphatidylethanolamine (PtdEtn) but not phosphatidylcholine (PtdCho) probes from its milieu. Consistently, the drug analogs of PtdCho are broadly ineffective in the parasite culture. NBD-PtdSer imported to the parasite interior is decarboxylated to NBD-PtdEtn, while the latter is not methylated to yield PtdCho, which confirms the expression of PtdSer decarboxylase but a lack of PtdEtn methyltransferase activity and suggests a role of exogenous lipids in membrane biogenesis of T. gondii . Flow cytometric quantitation of NBD-probes endorsed the selectivity of phospholipid transport and revealed a dependence of the process on energy and protein. Accordingly, our further work identified five P4-ATPases ( Tg P4-ATPase1-5), all of which harbor the signature residues and motifs required for phospholipid flipping. Of the four proteins expressed during the lytic cycle, Tg P4-ATPase1 is present in the apical plasmalemma; Tg P4-ATPase3 resides in the Golgi network along with its noncatalytic partner Ligand Effector Module 3 ( Tg Lem3), whereas Tg P4-ATPase2 and Tg P4-ATPase5 localize in the plasmalemma as well as endo/cytomembranes. Last but not least, auxin-induced degradation of Tg P4-ATPase1-3 impaired the parasite growth in human host cells, disclosing their crucial roles during acute infection. In conclusion, we show selective translocation of PtdEtn and PtdSer at the parasite surface and provide the underlying mechanistic and physiological insights in a model eukaryotic pathogen.

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“…Although the exact substrates a P4 ATPase transport differ, the substrate translocation path appears to have been conserved in all P4 ATPase studied so far ( Andersen et al, 2016 ; Huang et al, 2020 ). Like the Drs2 and ATP8A1 flippases, we found that the Dnf1 substrate translocation path is composed of TMH1-4 and TMH6 ( Figure 3A–D , Figure 3—figure supplement 1 ).…”

Section: Resultsmentioning

confidence: 99%

Transport mechanism of P4 ATPase phosphatidylcholine flippases

Bai

You

Jain

et al. 2020

eLife

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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.

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Conserved mechanism of phospholipid substrate recognition by the P4-ATPase Neo1 from Saccharomyces cerevisiae

Cited by 9 publications

References 54 publications

Transport Mechanism of Class-3 P4 ATPase Lipid Flippases

Transport Mechanism of Class-3 P4 ATPase Lipid Flippases

Aminoglycerophospholipid flipping and P4-ATPases in Toxoplasma gondii

Transport mechanism of P4 ATPase phosphatidylcholine flippases

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