Glycosylation limits forward trafficking of the tetraspan membrane protein PMP22

• Published in: The Journal of biological chemistry Vol. 296; p. 100719
• Main Authors: Marinko, Justin T., Wright, Madison T., Schlebach, Jonathan P., Clowes, Katherine R., Heintzman, Darren R., Plate, Lars, Sanders, Charles R.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 01-01-2021; American Society for Biochemistry and Molecular Biology
• Subjects: Charcot–Marie–Tooth disease (CMTD); ER quality control; N-linked glycosylation; peripheral myelin protein 22 (PMP22); peripheral neuropathy; trafficking; CMTD; ER quality control; Charcot–Marie–Tooth disease (CMTD); CNX; peripheral neuropathy; co-IP; gRNA; DMEM; PNS; FBS; EMC; trafficking; ERQC; RSC; GO; ER; ERAD; OST; RER1; PMP22; TM; TMT; peripheral myelin protein 22 (PMP22); N-linked glycosylation; PM; Charcot-Marie-Tooth disease (CMTD)
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/j.jbc.2021.100719

Peripheral myelin protein 22 (PMP22) folds and trafficks inefficiently, with only 20% of newly expressed protein trafficking to the cell surface. This behavior is exacerbated in many of the mutants associated with Charcot–Marie–Tooth disease, motivating further study. Here we characterized the role of N-glycosylation in limiting PMP22 trafficking. We first eliminated N-glycosylation using an N41Q mutation, which resulted in an almost 3-fold increase in trafficking efficiency of wildtype (WT) PMP22 and a 10-fold increase for the severely unstable L16P disease mutant in HEK293 cells, with similar results in Schwann cells. Total cellular levels were also much higher for the WT/N41Q mutant, although not for the L16P/N41Q form. Depletion of oligosaccharyltransferase OST-A and OST-B subunits revealed that WT PMP22 is N-glycosylated posttranslationally by OST-B, whereas L16P is cotranslationally glycosylated by OST-A. Quantitative proteomic screens revealed similarities and differences in the interactome for WT, glycosylation-deficient, and unstable mutant forms of PMP22 and also suggested that L16P is sequestered at earlier stages of endoplasmic reticulum quality control. CRISPR knockout studies revealed a role for retention in endoplasmic reticulum sorting receptor 1 (RER1) in limiting the trafficking of all three forms, for UDP-glucose glycoprotein glucosyltransferase 1 (UGGT1) in limiting the trafficking of WT and L16P but not N41Q, and calnexin (CNX) in limiting the trafficking of WT and N41Q but not L16P. This work shows that N-glycosylation is a limiting factor to forward trafficking PMP22 and sheds light on the proteins involved in its quality control.