Fig. 3

Construction of a comprehensive, predictive raft partitioning model. a TMD length and palmitoylation have been previously identified as determinants of raft partitioning. These have been formalized together with the surface area model from Fig. 2 into a comprehensive description of raft partitioning for TMDs. b Using parameters derived from published data and Fig. 2, raft affinity predicted from the tripartite model (ΔG_raft,pred) shows excellent agreement with measured values (ΔG_raft,app) for all tested TMDs (i.e., the constructs from Fig. 2a plus 4 palmitoylation mutants and 4 TMD truncations) without any floating fit parameters. c The model was used to predict raft affinity for the 729 single-pass PM proteins in the human proteome, from which three were chosen as representative raft-preferring proteins and two as non-raft preferring. d The partitioning of constructs containing TMDs from these five proteins followed predicted trends, with all three raft-predicted TMDs (CD4, LIME, PAG) partitioning efficiently to the raft domains, while both predicted non-raft proteins (LAX and LDLR) showed minimal raft partitioning. Vesicles in images are 5–10 μm in diameter. e-g The characteristics determining the raft partitioning of these constructs were dissected by mutating the TMDs to modify palmitoylation, TMD length, and ASA. e Mutating either of two palmitoylation sites reduced raft partitioning for LAT and PAG; f truncating the TMD length by 6 amino acids reduced raft partitioning for LAT, PAG, and LIME (but not LIME-Δ6exo); and g increasing ASA reduced raft partitioning for PAG and LIME, while decreasing ASA significantly increased raft partitioning for LDLR. Average±SD for 3–5 independent trials, each with > 10 vesicles/condition; *p<0.05; **p<0.01; ***p<0.001, one-way ANOVA. Sequences and partitioning values for all variants are given in Supplementary Tables 3, 4