Herrera Sara Abad, Justesen Bo Højen, Dieudonné Thibaud, Montigny Cédric, Nissen Poul, Lenoir Guillaume, Günther Pomorski Thomas
Department of Molecular Biochemistry, Faculty of Chemistry and Biochemistry, Ruhr University Bochum, Bochum, Germany.
Université Paris-Saclay, CEA, CNRS, Institute for Integrative Biology of the Cell (I2BC), Gif-sur-Yvette, France.
Protein Sci. 2023 Dec 8;33(3):e4855. doi: 10.1002/pro.4855.
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.
与Cdc50亚基结合的P4 - ATP酶是脂质翻转酶，它们将ATP水解与脂质转运偶联至膜的细胞质小叶，以产生脂质不对称性。这种定向转运已被证明有助于晚期分泌途径中的囊泡形成。一些翻转酶受自身抑制区域调节，这些区域可因蛋白激酶介导的磷酸化以及可能因胞质蛋白的结合而不稳定。此外，脂质与翻转酶的结合也可能诱导这些转运蛋白活性所需的构象变化。在这里，我们研究了磷脂酰肌醇 - 4 - 磷酸（PI4P）和末端自身抑制尾巴对酵母脂质翻转酶Drs2 - Cdc50的脂质翻转活性的作用。通过在与Cdc50亚基的1:1复合物中功能重建Drs2的全长和截短形式，我们提供了令人信服的证据，即脂质翻转酶活性仅在截短的Drs2变体中检测到，并且依赖于磷酸肌醇PI4P的存在。这些发现突出了磷酸肌醇作为脂质辅因子在Drs2 - Cdc50翻转酶调节脂质转运中的关键作用。
