Abstracts
Tools for measuring the compositional and biophysical paralipidome of cell membrane proteins
Ilya Levental
University of Virginia, Levental Lab of Membrane Biology, 1340 Jefferson Park Avenue, Charlottesville, VA 22903, USA, il2sy@virginia.edu
Membrane proteins constitute >30% of the mammalian proteome and 60% of all drug targets, localizing a major fraction of cellular bioactivity at membrane interfaces. Such proteins are potentially solvated by hundreds of distinct lipid subtypes with a range of physicochemical properties that can influence protein activity. Proteins have been hypothesized to selectively recruit local lipid environments, termed paralipidomes, that are chemically and physically distinct from the bulk membrane. While bulk membrane compositions and properties have been widely measured, the local lipid environments solvating membrane proteins have rarely been measured. To address this gap, we have developed tools for measuring the compositional and biophysical paralipidomes in live cells. For biophysical paralipidomes, we deployed HaloTags to covalently modify membrane proteins with environment-sensitive probes and thereby reveal the biophysical properties of various protein native paralipidomes in live cells. We use this modular approach to reveal asymmetry in local lipid packing between the inner and outer plasma membrane leaflets and how they are affected by lipid perturbations. We also observe distinct physical environments surrounding raft and non-raft protein markers, representing direct measurements of nanoscopic raft domains in live cells. For compositional paralipidomes, proteins are isolated with their native surrounding lipidomes using copolymeric nanodiscs, which are then analyzed by mass spectrometry. This approach reveals enrichment of specific lipid subtypes around GPCRs versus single-pass signaling adaptors. Thus, a modular toolbox enables straightforward measurements of local lipid nano-environments around membrane proteins and reveals layers of membrane organization in living cells.