1/11/2024 0 Comments Nih gag order![]() ![]() Mutating these residues results in reduction of Gag membrane association and of virus release ( 5, – 7). First, most retroviral Gag proteins have a cluster of basic residues in MA that interact with acidic lipids in the inner leaflet ( 4). Prior work in the field has provided a number of important clues to how Gag binds to membranes. Gag exploits one or more mechanisms governing binding of peripheral proteins to membranes, which include electrostatic interactions, hydrophobic interaction, recognition of specific lipid headgroups, protein multimerization, sensitivity to phospholipid acyl chain compositions, and preference for membrane order ( 3). How Gag interacts with the inner leaflet of the PM and how the biophysical properties of the PM contribute to Gag assembly are not fully understood. Gag is synthesized in the cytosol and then is targeted to the PM by the N-terminal domain, MA. The retroviral structural protein Gag provides the primary driving force for virus assembly at the inner leaflet of the plasma membrane (PM), and Gag alone is sufficient to assemble into virus-like particles (VLPs) in cells ( 1) and also in vitro ( 2). ![]() We also demonstrate how to define GUV phase composition, which could serve as a tool in future studies of protein membrane interactions. In contrast, the cellular protein domain MARCKS and the PS sensor Evectin2 show preference for disordered membranes. 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. Consistent with previous work on RSV Gag, we report here that electrostatic interactions provide the primary driving force for RSV Gag membrane association. This study examined how membrane charge and membrane order influence Gag membrane association. Our understanding of how Gag interacts with the PM and how different membrane properties contribute to overall Gag assembly is incomplete. ![]() IMPORTANCE Retroviral particles assemble on the PM and bud from infected cells. 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. Surprisingly, the PS sensor Evectin2 bound to the PS-rich Ld domain with 10-fold greater affinity than to the PS-rich Lo domain. Consistent with pelleting data, the MARCKS peptide showed preference for the Ld domain. RSV Gag and mNG-KRn membrane association followed membrane charge, independent of membrane order. 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 bound well to membranes with saturated and unsaturated acyl chains, whereas the MARCKS peptide and Evectin2 preferentially bound to membranes with unsaturated acyl chains. We used an in vitro liposome-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. 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. The retroviral structural protein Gag binds to the inner leaflet of the plasma membrane (PM), and many cellular proteins do so as well. ![]()
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