A glycosylated type I membrane protein becomes cytosolic when peptide: N ‐glycanase is compromised

The human cytomegalovirus‐encoded glycoprotein US2 catalyzes proteasomal degradation of Class I major histocompatibility complex (MHC) heavy chains (HCs) through dislocation of the latter from the endoplasmic reticulum (ER) to the cytosol. During this process, the Class I MHC HCs are deglycosylated by an N ‐glycanase‐type activity. siRNA molecules designed to inhibit the expression of the light chain, β 2 ‐microglobulin, block the dislocation of Class I MHC molecules, which implies that US2‐dependent dislocation utilizes correctly folded Class I MHC molecules as a substrate. Here we demonstrate it is peptide: N ‐glycanase (PNGase or PNG1) that deglycosylates dislocated Class I MHC HCs. Reduction of PNGase activity by siRNA expression in US2‐expressing cells inhibits deglycosylation of Class I MHC HC molecules. In PNGase siRNA‐treated cells, glycosylated HCs appear in the cytosol, providing the first evidence for the presence of an intact N ‐linked type I membrane glycoprotein in the cytosol. N ‐glycanase activity is therefore not required for dislocation of glycosylated Class I MHC molecules from the ER.