Creation of an optogenetic membrane editor with ultralow background activity

Membranes play important biological roles in compartmentalization, transport, and signaling, but the study of their lipid constituents is challenging due to constant flux through their complex metabolic and interorganelle transport pathways. To overcome these challenges, we developed optogenetic membrane editors for spatiotemporally precise manipulation of the lipid composition of organelle membranes. In this chapter, we describe the design and validation of LOVPLD, an ultralow background optogenetic membrane editor that enables acute production of phosphatidic acid (PA) on organelle membranes of interest. LOVPLD comprises the insertion of a blue light-sensitive conformational switch, a LOV domain, into the sequence of phospholipase D (PLD), a phosphatidic acid-synthesizing enzyme, rendering PLD activity dependent upon blue light stimulation. We envision that the "LOVification" strategy described herein may be applicable to other lipid-modifying enzymes to create membrane editors for targeting different lipid classes.