Advances over the past 25 years have revealed much about how the structural properties of membranes and associated proteins are linked to the thermodynamics and kinetics of membrane protein (MP) folding. At the same time biochemical progress has outlined how cellular proteostasis networks mediate MP folding and manage misfolding in the cell. When combined with results from genomic sequencing, these studies have established paradigms for how MP folding and misfolding are linked to the molecular etiologies of a variety of diseases. This emerging framework has paved the way for the development of a new class of small molecule “pharmacological chaperones” that bind to and stabilize misfolded MP variants, some of which are now in clinical use. In this review, we comprehensively outline current perspectives on the folding and misfolding of integral MPs as well as the mechanisms of cellular MP quality control. Based on these perspectives, we highlight new opportunities for innovations that bridge our molecular understanding of the energetics of MP folding with the nuanced complexity of biological systems. Given the many linkages between MP misfolding and human disease, we also examine some of the exciting opportunities to leverage these advances to address emerging challenges in the development of therapeutics and precision medicine.

Reconstitution of the PMP22 protein into lipid bilayers results in membrane assemblies that share common features with myelin.

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 S-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.

Charcot-Marie-Tooth disease (CMT) is a neuropathy of the peripheral nervous system that afflicts ~1:2500 people. The most common form of this disease (CMT1A, 1:4000) is associated with duplication of chromosome fragment 17p11.2-12, which results in a third wild-type PMP22 allele. In rodent models overexpressing the peripheral myelin protein 22 (PMP22) protein and in dermal fibroblasts from patients with CMT1A, PMP22 aggregates have been observed. This suggests that overexpression of PMP22 under CMT1A conditions overwhelms the endoplasmic reticulum (ER) quality control, leading to formation of cytotoxic aggregates. In this work, we used a single-cell flow-cytometry trafficking assay to quantitatively examine the relationship between PMP22 expression and trafficking efficiency in individual cells. We observed that as expression of wild-type or disease variants of PMP22 is increased, the amount of intracellular PMP22 increases to a greater extent than the amount of surface-trafficked protein. This was true for both transiently transfected cells as well as PMP22 stable expressing cells. Our results support the notion that overexpression of PMP22 in CMT1A leads to a disproportionate increase in misfolding and mis-trafficking of PMP22, which is likely a contributor to disease pathology and progression.

The ordered environment of membrane rafts is thought to exclude many transmembrane proteins. Nevertheless, some multi-pass transmembrane proteins have been proposed to partition into ordered domains. Here, giant plasma membrane vesicles (GPMVs) were employed to quantitatively show that the tetraspan peripheral myelin protein 22 (PMP22) exhibits a pronounced preference for, promotes the formation of, and stabilizes ordered membrane domains. Neither S-palmitoylation of PMP22 nor its putative cholesterol binding motifs are required for partitioning to ordered domains. In contrast, disruption of its unusual first transmembrane helix (TM1) reduced ordered phase preference. Charcot-Marie-Tooth disease-causing mutations that destabilize PMP22 also reduced or eliminated this preference in favor of the disordered phase. These studies demonstrate PMP22’s ordered phase preference derives both from the distinctive properties of TM1 and global structural features associated with its transmembrane domain, providing a first glimpse at the structural factors that promote raft partitioning for multi-pass proteins.Significance StatementThe preferential partitioning of single span membrane proteins for the ordered phase of ordered/disordered phase-separated membranes is now reasonably well understood, but little is known about this phase preferences of multi-pass membrane proteins. Here, it is shown that the disease-linked tetraspan integral membrane protein, PMP22, displays a pronounced preference to partition into the ordered phase, a preference that is reversed by disease mutations. This phase preference may be related to the role of PMP22 in cholesterol homeostasis in myelinating Schwann cells, a role that is also known to be disrupted under conditions of CMTD peripheral neuropathy caused by pmp22 mutations.