Direct evidence of lipid transport by the Drs2-Cdc50 flippase upon truncation of its terminal regions

Sara Abad Herrera; Bo Højen Justesen; Thibaud Dieudonné; Cédric Montigny; Poul Nissen; Guillaume Lenoir; Thomas Günther Pomorski

doi:10.1002/pro.4855

Direct evidence of lipid transport by the Drs2-Cdc50 flippase upon truncation of its terminal regions

Sara Abad Herrera, Bo Højen Justesen, Thibaud Dieudonné, Cédric Montigny, Poul Nissen, Guillaume Lenoir^*, Thomas Günther Pomorski^*

^*Corresponding author for this work

Research output: Contribution to journal/Conference contribution in journal/Contribution to newspaper › Journal article › Research › peer-review

Abstract

P4-ATPases in complex with Cdc50 subunits are lipid flippases that couple ATP hydrolysis with lipid transport to the cytoplasmic leaflet of membranes to create lipid asymmetry. Such vectorial transport has been shown to contribute to vesicle formation in the late secretory pathway. Some flippases are regulated by autoinhibitory regions that can be destabilized by protein kinase-mediated phosphorylation and possibly by binding of cytosolic proteins. In addition, the binding of lipids to flippases may also induce conformational changes required for the activity of these transporters. Here, we address the role of phosphatidylinositol-4-phosphate (PI4P) and the terminal autoinhibitory tails on the lipid flipping activity of the yeast lipid flippase Drs2–Cdc50. By functionally reconstituting the full-length and truncated forms of Drs2 in a 1:1 complex with the Cdc50 subunit, we provide compelling evidence that lipid flippase activity is exclusively detected for the truncated Drs2 variant and is dependent on the presence of the phosphoinositide PI4P. These findings highlight the critical role of phosphoinositides as lipid co-factors in the regulation of lipid transport by the Drs2–Cdc50 flippase.

Original language	English
Article number	e4855
Journal	Protein Science
Volume	33
Issue	3
Pages (from-to)	e4855
ISSN	0961-8368
DOIs	https://doi.org/10.1002/pro.4855
Publication status	Published - Mar 2024

Keywords

P4-ATPase
lipid flippase
membrane transporter
phosphoinositides
reconstitution

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@article{6a9e18d1064348439885689236bd8a02,

title = "Direct evidence of lipid transport by the Drs2-Cdc50 flippase upon truncation of its terminal regions",

abstract = "P4-ATPases in complex with Cdc50 subunits are lipid flippases that couple ATP hydrolysis with lipid transport to the cytoplasmic leaflet of membranes to create lipid asymmetry. Such vectorial transport has been shown to contribute to vesicle formation in the late secretory pathway. Some flippases are regulated by autoinhibitory regions that can be destabilized by protein kinase-mediated phosphorylation and possibly by binding of cytosolic proteins. In addition, the binding of lipids to flippases may also induce conformational changes required for the activity of these transporters. Here, we address the role of phosphatidylinositol-4-phosphate (PI4P) and the terminal autoinhibitory tails on the lipid flipping activity of the yeast lipid flippase Drs2–Cdc50. By functionally reconstituting the full-length and truncated forms of Drs2 in a 1:1 complex with the Cdc50 subunit, we provide compelling evidence that lipid flippase activity is exclusively detected for the truncated Drs2 variant and is dependent on the presence of the phosphoinositide PI4P. These findings highlight the critical role of phosphoinositides as lipid co-factors in the regulation of lipid transport by the Drs2–Cdc50 flippase.",

keywords = "P4-ATPase, lipid flippase, membrane transporter, phosphoinositides, reconstitution",

author = "Herrera, {Sara Abad} and Justesen, {Bo H{\o}jen} and Thibaud Dieudonn{\'e} and C{\'e}dric Montigny and Poul Nissen and Guillaume Lenoir and Pomorski, {Thomas G{\"u}nther}",

year = "2024",

month = mar,

doi = "10.1002/pro.4855",

language = "English",

volume = "33",

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journal = "Protein Science",

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Direct evidence of lipid transport by the Drs2-Cdc50 flippase upon truncation of its terminal regions. / Herrera, Sara Abad; Justesen, Bo Højen; Dieudonné, Thibaud et al.
In: Protein Science, Vol. 33, No. 3, e4855, 03.2024, p. e4855.

Research output: Contribution to journal/Conference contribution in journal/Contribution to newspaper › Journal article › Research › peer-review

TY - JOUR

T1 - Direct evidence of lipid transport by the Drs2-Cdc50 flippase upon truncation of its terminal regions

AU - Herrera, Sara Abad

AU - Justesen, Bo Højen

AU - Dieudonné, Thibaud

AU - Montigny, Cédric

AU - Nissen, Poul

AU - Lenoir, Guillaume

AU - Pomorski, Thomas Günther

PY - 2024/3

Y1 - 2024/3

N2 - P4-ATPases in complex with Cdc50 subunits are lipid flippases that couple ATP hydrolysis with lipid transport to the cytoplasmic leaflet of membranes to create lipid asymmetry. Such vectorial transport has been shown to contribute to vesicle formation in the late secretory pathway. Some flippases are regulated by autoinhibitory regions that can be destabilized by protein kinase-mediated phosphorylation and possibly by binding of cytosolic proteins. In addition, the binding of lipids to flippases may also induce conformational changes required for the activity of these transporters. Here, we address the role of phosphatidylinositol-4-phosphate (PI4P) and the terminal autoinhibitory tails on the lipid flipping activity of the yeast lipid flippase Drs2–Cdc50. By functionally reconstituting the full-length and truncated forms of Drs2 in a 1:1 complex with the Cdc50 subunit, we provide compelling evidence that lipid flippase activity is exclusively detected for the truncated Drs2 variant and is dependent on the presence of the phosphoinositide PI4P. These findings highlight the critical role of phosphoinositides as lipid co-factors in the regulation of lipid transport by the Drs2–Cdc50 flippase.

AB - P4-ATPases in complex with Cdc50 subunits are lipid flippases that couple ATP hydrolysis with lipid transport to the cytoplasmic leaflet of membranes to create lipid asymmetry. Such vectorial transport has been shown to contribute to vesicle formation in the late secretory pathway. Some flippases are regulated by autoinhibitory regions that can be destabilized by protein kinase-mediated phosphorylation and possibly by binding of cytosolic proteins. In addition, the binding of lipids to flippases may also induce conformational changes required for the activity of these transporters. Here, we address the role of phosphatidylinositol-4-phosphate (PI4P) and the terminal autoinhibitory tails on the lipid flipping activity of the yeast lipid flippase Drs2–Cdc50. By functionally reconstituting the full-length and truncated forms of Drs2 in a 1:1 complex with the Cdc50 subunit, we provide compelling evidence that lipid flippase activity is exclusively detected for the truncated Drs2 variant and is dependent on the presence of the phosphoinositide PI4P. These findings highlight the critical role of phosphoinositides as lipid co-factors in the regulation of lipid transport by the Drs2–Cdc50 flippase.

KW - P4-ATPase

KW - lipid flippase

KW - membrane transporter

KW - phosphoinositides

KW - reconstitution

U2 - 10.1002/pro.4855

DO - 10.1002/pro.4855

M3 - Journal article

C2 - 38063271

SN - 0961-8368

VL - 33

SP - e4855

JO - Protein Science

JF - Protein Science

IS - 3

M1 - e4855

ER -

Direct evidence of lipid transport by the Drs2-Cdc50 flippase upon truncation of its terminal regions

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Keywords

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