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 multipass 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 multipass TMP partitioning to ordered membrane domains. Across a set of 24 structurally and functionally diverse multipass 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 proteo lipid 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 cholesterol-mediated 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 multipass transmembrane proteins.

Figure 1 –. GPMVs as a biomimetic model of lipid raft affinity for PM proteins.

(A) Example of quantification of raft affinity for a raft-preferring protein. A protein of interest (LAT= TMD of Linker for Activation of T-Cells, red) is expressed in cells, which are stained with a lipid dye with a known phase preference (F-DiO, green, non-raft). The intensity of the protein in the raft versus non-raft phase yields raft partition coefficient (K_{p, raft}). LAT has K_{p, raft} > 1, and is thus a raft phase preferring protein. (B) Example of quantification of a non-raft preferring protein. Same principle as panel A, but with the TMD of LDLR, which is largely excluded from the raft phase. (C) Comparison of raft affinity for several single pass proteins and three multi-pass TMPs. Single TMD raft affinity is in good agreement with predictions , whereas multi-pass proteins that have been previously associated with rafts have minimal raft phase affinity. Average +/− st. dev. for at least 10 vesicles for each protein, representative of three independent trials.

Figure 2 –. Almost all multi-pass TMPs have low raft affinity in GPMVs.

Shown are the raft affinity of multi-pass TMPs with varying number of TMHs. Raft affinity is shown in log scale, with 0 representing equal partitioning between the phases and positive and negative values representing enrichment in or depletion from the raft phase, respectively. Included in each panel is the value for LAT (a raft-preferring single pass TMD, black) and LDLR (a non-raft TMD, white). (A) 2-TM proteins. Three homologs of MOG from different species (h:human; m:mouse; r:rat) and both isoforms (alpha and beta) of the human protein are excluded from rafts, as is the unrelated CD36. (B) Two 4-TM proteins are highly raft-preferring, namely MAL and PLP. In contrast, several other members of the MARVEL family (BENE, MAL2, occludin, and synaptophysin) and other tetraspanin family proteins (CD9, CD81, CD82), as well as the neuronal membrane glycoprotein (GPM6a) are almost completely excluded. Two analogs of MAL (c:canine: h:human) were tested and both had significantly greater raft affinity than LAT. (C) All tested 6-TM and 7-TM proteins are raft excluded. Several proteins from the TRP family (TRPA1, TRPM8, TRPV1), Adenylyl Cyclase 9 (AC9), and G Protein-Coupled Receptors (DRD1, β2AR) were not significantly different from LDLR. (D) All tested proteins with 10 or more TMDs - PM scramblase TMEM16F and several transporters (NKCC1, KCNQ1 and GAT1) - were raft excluded. Data points represent the means of individual experiments of >10 vesicles each.

Figure 3 –. LIRW peptide is important for MAL ER exit but not raft affinity.

(A) Schematics for MAL (red) with LIRW motif close to the cytosolic C-terminal and PLP (green) with numerous palmitoylation at cytosolic loops. (B) Images of GPMVs containing cMAL-RFP (MAL), PLP-GFP (PLP) and trLAT, with their respective complementary non-raft marker, showing that all three proteins prefer the raft phase. (C) Raft affinity (log K_{p, raft}) measurements show MAL has higher raft affinity than PLP and LAT, which have similar raft affnity. D) Representative images for HEK-293 cells transfected with cMAL and C-terminal mutants. Wild-type cMAL (wt) accumulates in the PM with some punctate structures while single point mutations of the LIRW motif lead to clear accumulation in the ER. (E) Mutations in LIRW motif do not change lipid raft affinity of the protein. Both mutants, L175A and W178S, showed a retention in the ER, but the minimal amount of protein present in PM still partitioned into the raft phase in GPMVs. Scale bar corresponds to 10 μm. ***p<0.001 by t-test

Figure 4 –. MAL displaces PLP from raft phase.

(A) Single transfected PLP and MAL both have preference for raft phase (non-raft labeled with F-DiO, white). Co-transfection reduces PLP raft preference to approximately equal partitioning between phases, but does not significantly affect MAL. (B) PLP partitioning is unaffected by co-transfection with LAT. (C) Raft partitioning of MAL (open symbols) and PLP (closed symbols) as a function of co-transfection (triangles) compared with single transfected (circles). PLP raft affinity is significantly reduced by co-transfection with MAL, (D) but not by the co-transfection with LAT. Partitioning in panel D shown as normalized by PLP alone. Bars represent average +/− s.e.m.; circles represent means of individual experiments (>10 vesicles each). **p<0.01 by t-test.

Figure 5 –. MAL and PLP may compete for cholesterol.

(A) Cholesterol depletion does not significantly affect MAL partitioning, which still enriches in raft phase upon cholesterol depletion with MβCD. (B) PLP partitioning is reduced by cholesterol depletion, becoming approximately homogeneously distributed between phases. (C) Cholesterol depletion severely reduces PLP raft affinity when co-expressed with MAL. Under this condition, PLP is excluded from rafts domains and co-enriches with the non-raft marker (F-DiD: white). (D) Quantifications of all conditions. (E) LAT partitioning is not affected by MβCD treatment. Bars represent average +/− s.e.m.; circles represent means of individual experiments (>10 vesicles each). *p<0.1, **p<0.01, ***p<0.001 by t-test. (F) PLP partitioning negatively correlates with the abundance of MAL. (i.e. relative intensity of MAL to PLP). (F) The effect is enhanced by cholesterol depletion with MβCD.

Figure 6 –. Sphingolipids are required for PLP raft enrichment.

(A) MAL raft affinity is not significantly affected by sphingolipid depletion. (B) In contrast, PLP enriched in non-raft phrase upon myriocin treatment. (C) Quantification of myriocin-dependent partitioning. (D) Myriocin effect on PLP is not affected by co-transfection with MAL. Bars represent average +/− s.e.m.; circles represent means of individual experiments (>10 vesicles each). **p<0.01.