Marinko, J. T. | Search bioRxiv

bioRxiv2020

Glycosylation Limits Forward Trafficking of the Tetraspan Membrane Protein PMP22

Peripheral myelin protein 22 (PMP22) folds and traffics inefficiently, a phenomenon closely related to the mechanisms by which this tetraspan membrane protein causes Charcot-Marie-Tooth disease (CMTD). We report that elimination of N-glycosylation results in a 3-fold increase in the cell surface trafficking of wild type (WT) PMP22 and a 10-fold increase in trafficking of the unstable L16P disease mutant form. Studies of the interactions of PMP22 with oligosaccharyltransferases A and B as well as quantitative proteomic experiments established that critical endoplasmic reticulum (ER) quality control decisions occur earlier in the biogenesis to cell surface trafficking pathway for the L16P mutant than for WT. CRISPR knock-out cell lines for ER proteins calnexin, RER1, and UGGT1 illuminated the role of each protein in glycosylation dependent and independent surface trafficking of WT PMP22, as well as for a series of disease mutants of varying folding stabilities. One Sentence SummaryN-linked glycosylation was seen to dramatically limit the cell surface trafficking of PMP22, with some key quality control factors in PMP22 biogenesis being identified.

cell biology↗

bioRxiv2020

Peripheral Myelin Protein 22 Preferentially Partitions into Ordered Phase Membrane Domains

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 PMP22s 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.

biophysics↗