Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, Toyama, Japan.
Graduate School of Pharmaceutical Sciences, Kyoto University, Sakyo-ku, Kyoto, Japan.
Biophys J. 2019 Jan 8;116(1):92-103. doi: 10.1016/j.bpj.2018.11.3131. Epub 2018 Dec 4.
Sec14, the major yeast phosphatidylcholine (PC)/phosphatidylinositol (PI) transfer protein (PITP), coordinates PC and PI metabolism to facilitate an appropriate and essential lipid signaling environment for membrane trafficking from trans-Golgi membranes. The Sec14 PI/PC exchange cycle is essential for its essential biological activity, but fundamental aspects of how this PITP executes its lipid transfer cycle remain unknown. To address some of these outstanding issues, we applied time-resolved small-angle neutron scattering for the determination of protein-mediated intervesicular movement of deuterated and hydrogenated phospholipids in vitro. Quantitative analysis by small-angle neutron scattering revealed that Sec14 PI- and PC-exchange activities were sensitive to both the lipid composition and curvature of membranes. Moreover, we report that these two parameters regulate lipid exchange activity via distinct mechanisms. Increased membrane curvature promoted both membrane binding and lipid exchange properties of Sec14, indicating that this PITP preferentially acts on the membrane site with a convexly curved face. This biophysical property likely constitutes part of a mechanism by which spatial specificity of Sec14 function is determined in cells. Finally, wild-type Sec14, but not a mixture of Sec14 proteins specifically deficient in either PC- or PI-binding activity, was able to effect a net transfer of PI or PC down opposing concentration gradients in vitro.
Sec14 是主要的酵母磷脂酰胆碱（PC）/磷脂酰肌醇（PI）转位蛋白（PITP），它协调 PC 和 PI 代谢，以促进适当和必需的脂质信号环境，从而促进从反式高尔基体膜的膜运输。Sec14 的 PI/PC 交换循环对于其基本的生物活性是必需的，但这种 PITP 如何执行其脂质转移循环的基本方面仍然未知。为了解决其中的一些悬而未决的问题，我们应用时间分辨小角中子散射来确定体外氘化和氢化磷脂在蛋白介导的囊泡间运动。小角中子散射的定量分析表明，Sec14 的 PI 和 PC 交换活性对膜的脂质组成和曲率都很敏感。此外，我们报告这两个参数通过不同的机制调节脂质交换活性。增加膜曲率促进 Sec14 的膜结合和脂质交换特性，表明该 PITP 优先作用于具有凸面曲率的膜位点。这种生物物理特性可能构成了 Sec14 功能空间特异性在细胞中确定的部分机制。最后，野生型 Sec14，但不是专门缺乏 PC 或 PI 结合活性的 Sec14 蛋白混合物，能够在体外有效地将 PI 或 PC 沿着相反的浓度梯度进行净转移。
