Fig. 1

Raft-targeting features distributed over the TMD of LAT. a Sequences of TMDs used to determine the localization of the raft-targeting feature. Mutations were made to either the entire TMD sequence (trLAT-allL) or to individual amino acid quartets (e.g. 1pL). b  Example of quantification of raft partitioning in GPMVs. A protein of interest (trLAT; red) is expressed in cells, which are then stained with a lipid dye (F-DiO; green) with known phase preference (non-raft phase for F-DiO). The dye is used to identify the non-raft phase in GPMVs, and the relative fluorescence intensity of the protein in the raft versus non-raft phase gives the raft partition coefficient, K_p,raft. Mutating all TMD residues to Leu (trLAT-allL) decreases the raft affinity relative to the native LAT TMD (trLAT-wt). Vesicles in images are 5–10 μm in diameter. c The TMD was divided into 6 parts, that were mutated individually to Leu to identify the location of the raft-targeting features. None of these partial mutations reproduced the lack of raft affinity of the all-Leu construct, suggesting a distributed feature responsible for the raft affinity of the LAT TMD. d The 16 core residues of the LAT TMD were randomized to create trLAT-scr. This construct partitioned at parity with the wild-type TMD, suggesting amino acid properties, rather than sequence, as the key determinant of raft affinity. Average±SD for 3–5 independent trials, each with > 10 vesicles/condition; **p<0.01; ***p<0.001, one-way ANOVA. Sequences and partitioning values for all variants are given in Supplementary Table 1