Effects of Membrane Charge and Order on Membrane Binding of the Retroviral Structural Protein Gag.
Effects of Membrane Charge and Order on Membrane Binding of the Retroviral Structural Protein Gag.
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膜电荷和顺序对逆转录病毒结构蛋白 Gag 膜结合的影响。
DOI：
10.1128/jvi.01102-16
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发表时间：
2016
影响因子：
5.4
通讯作者：
Vogt,VolkerM
中科院分区：
文献类型：
--
作者：
Wen,Yi;Dick,RobertA;Feigenson,GeraldW;Vogt,VolkerM
逆转录病毒结构蛋白Gag与质膜（PM）的内小叶结合，许多细胞蛋白也是如此。我们使用劳斯肉瘤病毒（RSV）Gag与膜传感器一起研究外周蛋白膜结合的原理，包括静电，磷脂头基的特异性识别，对磷脂酰基链组合物的敏感性，对膜顺序的偏好和蛋白质多聚化。我们使用体外脂质体沉淀法来测试Gag、已充分表征的MARCKS肽、一系列荧光静电传感器蛋白（mNG-KRn）和特异性磷脂酰丝氨酸（PS）结合蛋白Evectin 2的蛋白膜结合特性。RSV Gag和mNG-KRn与具有饱和和不饱和酰基链的膜结合良好，而MARCKS肽和Evectin 2优先与具有不饱和酰基链的膜结合。为了进一步区分Gag膜结合的主要驱动力是否是静电相互作用或膜顺序的偏好，我们测量了蛋白质结合到在无序（Ld）和有序（Lo）相中含有相同PS浓度的巨单层囊泡（GUV）。RSV Gag和mNG-KRn膜结合遵循膜电荷，与膜顺序无关。与沉淀数据一致，MARCKS肽显示出对Ld结构域的偏好。令人惊讶的是，PS传感器Evectin 2以比富含PS的Lo结构域大10倍的亲和力结合富含PS的Ld结构域。总之，我们发现RSV Gag对膜顺序没有偏好，而具有报道的膜穿透结构域的蛋白质对无序的膜有偏好。重要的是逆转录病毒颗粒在PM上组装并从感染的细胞中出芽。我们对Gag如何与PM相互作用以及不同的膜性质如何有助于整体Gag组装的理解是不完整的。本研究探讨了如何膜电荷和膜的顺序影响加格膜协会。与以前对RSV Gag的工作一致，我们在这里报告静电相互作用为RSV Gag膜缔合提供了主要驱动力。使用相分离的GUV与已知的脂质组成的Ld和Lo相，我们证明了第一次，RSV Gag是敏感的膜电荷，但不是膜顺序。相反，细胞蛋白结构域MARCKS和PS传感器Evectin 2显示出对无序膜的偏好。我们还演示了如何定义GUV相组成，这可以作为一个工具，在未来的研究蛋白质膜相互作用。
The retroviral structural protein Gag binds to the inner leaflet of the plasma membrane (PM), and many cellular proteins do so as well. We used Rous sarcoma virus (RSV) Gag together with membrane sensors to study the principles governing peripheral protein membrane binding, including electrostatics, specific recognition of phospholipid headgroups, sensitivity to phospholipid acyl chain compositions, preference for membrane order, and protein multimerization. We used anin vitroliposome-pelleting assay to test protein membrane binding properties of Gag, the well-characterized MARCKS peptide, a series of fluorescent electrostatic sensor proteins (mNG-KRn), and the specific phosphatidylserine (PS) binding protein Evectin2. RSV Gag and mNG-KRn bound well to membranes with saturated and unsaturated acyl chains, whereas the MARCKS peptide and Evectin2 preferentially bound to membranes with unsaturated acyl chains. To further discriminate whether the primary driving force for Gag membrane binding is electrostatic interactions or preference for membrane order, we measured protein binding to giant unilamellar vesicles (GUVs) containing the same PS concentration in both disordered (Ld) and ordered (Lo) phases. RSV Gag and mNG-KRn membrane association followed membrane charge, independent of membrane order. Consistent with pelleting data, the MARCKS peptide showed preference for the Ld domain. Surprisingly, the PS sensor Evectin2 bound to the PS-rich Ld domain with 10-fold greater affinity than to the PS-rich Lo domain. In summary, we found that RSV Gag shows no preference for membrane order, while proteins with reported membrane-penetrating domains show preference for disordered membranes.IMPORTANCERetroviral particles assemble on the PM and bud from infected cells. Our understanding of how Gag interacts with the PM and how different membrane properties contribute to overall Gag assembly is incomplete. This study examined how membrane charge and membrane order influence Gag membrane association. Consistent with previous work on RSV Gag, we report here that electrostatic interactions provide the primary driving force for RSV Gag membrane association. Using phase-separated GUVs with known lipid composition of the Ld and Lo phases, we demonstrate for the first time that RSV Gag is sensitive to membrane charge but not membrane order. In contrast, the cellular protein domain MARCKS and the PS sensor Evectin2 show preference for disordered membranes. We also demonstrate how to define GUV phase composition, which could serve as a tool in future studies of protein membrane interactions.