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Oligosaccharyltransferases (OSTs) mediate the en bloc transfer of N‐glycan intermediates onto the asparagine residue in glycosylation sequons (N‐X‐S/T, X≠P). These enzymes are typically heteromeric complexes composed of several membrane‐associated subunits, in which STT3 is highly conserved as a catalytic core. Metazoan organisms encode two STT3 genes (STT3A and STT3B) in their genome, resulting in the formation of at least two distinct OST isoforms consisting of shared subunits and complex specific subunits. The STT3A isoform of OST primarily glycosylates substrate polypeptides cotranslationally, whereas the STT3B isoform is involved in cotranslational and post‐translocational glycosylation of sequons that are skipped by the STT3A isoform. Here, we describe mutant constructs of monomeric enhanced green fluorescent protein (mEGFP), which are susceptible to STT3B‐dependent N‐glycosylation. The endoplasmic reticulum‐localized mEGFP (ER‐mEGFP) mutants contained an N‐glycosylation sequon at their C‐terminus and exhibited increased fluorescence in response to N‐glycosylation. Isoform‐specific glycosylation of the constructs was confirmed by using STT3A‐ or STT3B‐knockout cell lines. Among the mutant constructs that we tested, the ER‐mEGFP mutant containing the N185‐C186‐T187 sequon was the best substrate for the STT3B isoform in terms of glycosylation efficiency and fluorescence change. Our results suggest that the mutant ER‐mEGFP is useful for monitoring STT3B‐dependent post‐translocational N‐glycosylation in cells of interest, such as those from putative patients with a congenital disorder of glycosylation. [ABSTRACT FROM AUTHOR]

Glycosylphosphatidylinositol (GPI) anchoring of proteins is a conserved posttranslational modification in the endoplasmic reticulum (ER). Soon after GPI is attached, an acyl chain on the GPI inositol is removed by post-GPI attachment to proteins 1 (PGAP1), a GPI-inositol deacylase. This is crucial for switching GPI-anchored proteins (GPI-APs) from protein folding to transport states. We performed haploid genetic screens to identify factors regulating GPI-inositol deacylation, identifying seven genes. In particular, calnexin cycle impairment caused inefficient GPI-inositol deacylation. Calnexin was specifically associated with GPI-APs, dependent on N -glycan and GPI moieties, and assisted efficient GPI-inositol deacylation by PGAP1. Under chronic ER stress caused by misfolded GPI-APs, inositol-acylated GPI-APs were exposed on the cell surface. These results indicated that N -glycans participate in quality control and temporal ER retention of GPI-APs, ensuring their correct folding and GPI processing before exiting from the ER. Once the system is disrupted by ER stress, unprocessed GPI-APs become exposed on the cell surface., (© 2018 Liu et al.)