RT Journal Article SR Electronic T1 An inducible amphipathic α-helix mediates subcellular targeting and membrane binding of RPE65 JF Life Science Alliance JO Life Sci. Alliance FD Life Science Alliance LLC SP e202201546 DO 10.26508/lsa.202201546 VO 6 IS 1 A1 Uppal, Sheetal A1 Liu, Tingting A1 Galvan, Emily A1 Gomez, Fatima A1 Tittley, Tishina A1 Poliakov, Eugenia A1 Gentleman, Susan A1 Redmond, T Michael YR 2023 UL https://www.life-science-alliance.org/content/6/1/e202201546.abstract AB RPE65 retinol isomerase is an indispensable player in the visual cycle between the vertebrate retina and RPE. Although membrane association is critical for RPE65 function, its mechanism is not clear. Residues 107–125 are believed to interact with membranes but are unresolved in all RPE65 crystal structures, whereas palmitoylation at C112 also plays a role. We report the mechanism of membrane recognition and binding by RPE65. Binding of aa107–125 synthetic peptide with membrane-mimicking micellar surfaces induces transition from unstructured loop to amphipathic α-helical (AH) structure but this transition is automatic in the C112-palmitoylated peptide. We demonstrate that the AH significantly affects palmitoylation level, membrane association, and isomerization activity of RPE65. Furthermore, aa107–125 functions as a membrane sensor and the AH as a membrane-targeting motif. Molecular dynamic simulations clearly show AH-membrane insertion, supporting our experimental findings. Collectively, these studies allow us to propose a working model for RPE65-membrane binding, and to provide a novel role for cysteine palmitoylation.