Myelin-Associated MAL and PLP Are Unusual among Multipass Transmembrane Proteins in Preferring Ordered Membrane Domains

Abstract

Eukaryotic membranes can be partitioned into lipid-driven membrane microdomains called lipid rafts, which function to sort lipids and proteins in the plane of the membrane. As protein selectivity underlies all functions of lipid rafts, there has been significant interest in understanding the structural and molecular determinants of raft affinity. Such determinants have been described for lipids and single-spanning transmembrane proteins; however, how multi-pass transmembrane proteins (TMPs) partition between ordered and disordered phases has not been widely explored.Here we used cell-derived Giant Plasma Membrane Vesicles (GPMVs) to systematically measure multi-pass TMP partitioning to ordered membrane domains. Across a set of 24 structurally and functionally diverse multi-pass TMPs, the large majority (92%) had minimal raft affinity. The only exceptions were two myelin-associated four pass TMPs, Myelin and Lymphocyte protein (MAL) and ProteoLipid Protein (PLP). We characterized the potential mechanisms for their exceptional raft affinity and observed that PLP requires cholesterol and sphingolipids for optimal association with ordered membrane domains and that PLP and MAL appear to compete for cholesterolmediated raft affinity. These observations suggest broad conclusions about the composition of ordered membrane domains in cells and point to previously unrecognized drivers of raft affinity for multi-pass transmembrane proteins.

SARS-CoV-2 Requires Cholesterol for Viral Entry and Pathological Syncytia Formation

“…Given previous results using indirect detergent fractionation readouts (McBride and Machamer, 2010b;Petit et al, 2007), we were surprised to observe that SARS-CoV-2 spike does not partition strongly into GPMVs' dense, ordered phase (L o ) (Figure 6D), which is enriched for cholesterol and sphingolipids (Levental et al, 2009;. Moreover, in our cell fusion assay, treatment with the raft-disrupting drug myriocin, which depletes sphingolipids from the plasma membrane (Castello-Serrano et al, 2020), did not inhibit fusion (Figure 6F,G). Thus, SARS-CoV-2 spike protein appears to facilitate membrane-fusion in a manner that is dependent on palmitoylation of its uniquely cysteine-rich CTD, but through a mechanism unique from canonical membrane nanodomains, although we cannot rule out a discrepancy in lipid raft properties between GPMVs and living cells (Levental et al, 2020).…”

SARS-CoV-2 requires cholesterol for viral entry and pathological syncytia formation

“…Given previous results using indirect detergent fractionation readouts (McBride and Machamer, 2010b;Petit et al, 2007), we were surprised to observe that SARS-CoV-2 spike does not partition strongly into GPMVs' dense, ordered phase (L o ) (Figure 6D), which is enriched for cholesterol and sphingolipids (Levental et al, 2009;. Moreover, in our cell fusion assay, treatment with the raft-disrupting drug myriocin, which depletes sphingolipids from the plasma membrane (Castello-Serrano et al, 2020), did not inhibit fusion (Figure 6F,G). Thus, SARS-CoV-2 spike protein facilitates membrane-fusion in a manner that could be dependent on palmitoylation of its uniquely cysteine-rich CTD, but through a mechanism unique from canonical membrane nanodomains, although we cannot rule out a discrepancy in lipid raft properties between GPMVs and living cells (Levental et al, 2020).…”

Active cholesterol 20 years on

“…The partition of some integral plasma membrane proteins between liquid‐ordered and liquid‐disordered domains in a bilayer can vary with physiological as well as experimental conditions 29 . It is possible that cholesterol is not only integral to the formation of rafts but also that active cholesterol can drive certain proteins into ordered domains 184‐186 . The active cholesterol‐dependent behavior of some of the aforementioned proteins could then reflect such transitions.…”