Abstract
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.
Highlights
We focused on four potential mediators of peripheral myelin protein 22 (PMP22) trafficking: CNX, lectin mannose-binding protein 1 (LMAN1, known as endoplasmic reticulum (ER)–Golgi intermediate compartment 53 kDa protein— ERGIC53), UDP-glucose:glycoprotein glucosyltransferase 1 (UGGT1), and receptor 1 (RER1)
The present results suggest that RER1 functions as a glycosylation-independent Golgi-toER retrograde transporter for both WT and disease mutant forms of PMP22, a function that reflects a role for RER1 in preventing the escape of folding-immature proteins from the ER
It was seen that the molecular pathways of N-glycosylation are different for WT PMP22 and less stable forms
Summary
Hydrophilic environments [7]. Soluble proteins translocate through the pore to enter the ER lumen, while TM helices partition laterally into the ER membrane. Protein folding in the ER is under constant surveillance by the resident ER quality control network (ERQC) [1, 10, 11], which contains numerous folding sensors, chaperones, and other proteins, including those involved in ERAD and ERassociated autophagy. These proteins monitor, assist, and execute logic decisions as to whether to retain, degrade, or authorize exit of nascent proteins from the ER. Mutant forms of PMP22 are known to traffic even less efficiently than WT, consistent with the notion that peripheral neuropathies associated with these PMP22 variants are the consequence of defects in PMP22 trafficking
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