A nile red fluorescence assay for LC3 autophagy protein binding to lipid bilayers

Autophagy consists of the degradation and recycling of defective or aging cellular components. This process occurs in all eukaryotes, and starts by the formation of a double-bilayer structure known as the autophagosome. In humans, autophagosome generation requires, among others, the action of three homologous proteins, designated as LC3A, LC3B, and LC3C. These are amphipathic proteins, which can exist either in aqueous or in membranous environments. Quantification of LC3 binding to lipid bilayers is usually achieved by a rather cumbersome procedure, involving separation of bound and free forms by density gradient centrifugation and fractional analysis of the centrifuged samples. This paper describes a simple protein binding assay based on the fluorescence properties of Nile red. This solvatochromic probe has recently been applied to the study of lipid bilayer fluidity. A red/orange intensity ratio (ROIR) index, derived from the Nile red emission spectrum, was found useful in order to normalize the results [Sot et al. 2022, doi: https://doi.org/10.1016/j.molliq.2022.119874 ]. The current results show that LC3 protein binding to bilayers was accompanied by a protein-concentration-dependent decrease of ROIR, and there was a strict correlation between the bindings measured by ultracentrifugation and by fluorescence. ROIR decreased with LC3 concentrations following a hyperbolic curve and this allowed an estimation of the maximum decrease for each lipid composition. In agreement with previous observations, the presence of cardiolipin and ceramide in the bilayer markedly facilitated binding. Even if absolute values of binding cannot be obtained, the Nile red method may become of general use in the assay of amphipathic protein interaction with bilayers.