The ordered environment of cholesterol-rich membrane nanodomains is thought to exclude many transmembrane (TM) proteins. Nevertheless, some multispan helical transmembrane proteins have been proposed to partition into these environments. Here, giant plasma membrane vesicles (GPMVs) were employed to quantitatively show that the helical tetraspan peripheral myelin protein 22 (PMP22) exhibits a pronounced preference for, promotes the formation of, and stabilizes ordered membrane domains. Neither

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... palmitoylation of PMP22 nor its putative cholesterol binding motifs are required for this preference. In contrast, Charcot–Marie–Tooth disease-causing mutations that disrupt the stability of PMP22 tertiary structure reduce or eliminate this preference in favor of the disordered phase. These studies demonstrate that the ordered phase preference of PMP22 derives from global structural features associated with the folded form of this protein, providing a glimpse at the structural factors that promote raft partitioning for multispan helical membrane proteins.