Overcoming Fluorescence Loss in mEOS-based AAA+ Unfoldase Reporters Through Covalent Linkage

Recent work has demonstrated that the soluble photoconvertable fluorescent protein mEOS can be a reporter for AAA+ (ATPases Associated with diverse cellular Activities) unfoldase activity. Given that many AAA+ proteins process membrane proteins, we sought to adapt mEOS for use with membrane protein substrates. However, direct genetic fusion of mEOS to a membrane protein completely abolished fluorescence, severely limiting the utility of mEOS for studying AAA+ proteins. To circumvent this challenge, we separately purified mEOS and a AAA+ degron, covalently linked them via Sortase, and photoconverted the linked construct. This innovative approach preserves fluorescence and enables functional analysis, offering a broadly applicable platform for the study of membrane associated AAA+ proteins.