A new method for studying the orientation of membrane proteins in plants based on the release of a fluorescent protein tag by TEV protease

Dear Editor,The orientation of a membrane protein can provide important information about its interaction partners,function,and mechanism of action.In addition to traditional biochemical assays,new methods have been developed to reveal the orientation of membrane proteins.For example,computer programs have been developed to predict membrane protein topology but often have inaccuracies(Ott and Lingappa,2002;Schwacke et al.,2003).A widely used method is the fluorescence protease protection(FPP)assay(Lomize et al.,2006;Lorenz et al.,2008;White et al.,2015).However,it often fails to give good results for assays of plant membrane proteins.Here,we developed a simpler method based on release of a fluorescent protein tag(RFT)by the tobacco etch virus(TEV)protease to overcome the shortcomings of FPP and analyze the orientation of membrane proteins in plants.

Cis-interaction of ligands on a supported lipid bilayer afects their binding to cell adhesion receptors

Cell adhesion on a supported lipid bilayer(SLB) functionalized with ligand proteins is a widely-used cell-mimetic model for the study of the trans receptor-ligand interaction that mediates the adhesion, and has been shown to play an important role in unravelling the molecular players in cell adhesion. Experimental studies of such cell-SLB adhesion systems often assume that there is no cis interactions between the ligands. An important question remains whether the cis-interaction afects the trans receptor-ligand interaction. Using a statistical-mechanical model and Monte Carlo simulations with biologically relevant parameters, we find that the attractive cis-interaction of strength 1 kBT between adjacent ligands on the SLB can lead to an amplification of both the affinity and cooperativity of the receptor-ligand binding, thereby facilitating the phase separation within the adhering cell membrane. In contrast, the adhesion system is less sensitive to the repulsive cis-interaction between adjacent ligands on the SLB.Our results suggest that the ligand-ligand cis-interaction should be carefully considered in the cell-SLB adhesion experiments.