The deglycosylating enzyme, peptide:N-glycanase, acts on misfolded N-linked glycoproteins dislocated from the endoplasmic reticulum (ER) to the cytosol. Deglycosylation has been demonstrated to occur at the ER membrane and in the cytosol. However, the mechanism of PNGase association with the ER membrane was unclear, because PNGase lacked the necessary signal to facilitate its incorporation in the ER membrane, nor was it known to bind to an integral ER protein.Using HeLa cells, we have identified a membrane protein that associates with PNGase, thereby bringing it in close proximity to the ER and providing accessibility to dislocating glycoproteins. This protein, Derlin-1, has recently been shown to mediate retrotranslocation of misfolded glycoproteins. In this study we demonstrate that Derlin-1 interacts with the N-terminal domain of PNGase via its cytosolic C-terminus. Moreover, we find PNGase distributed in two populations; ER-associated and free in the cytosol, which suggests the deglycosylation process can proceed at either site depending on the glycoprotein substrate. INTRODUCTIONIn eukaryotes proteins are synthesized on membrane-bound ribosomes and undergo a folding process in the lumen of the endoplasmic reticulum (ER; Helenius, 2001, 2003). Misfolded or incompletely assembled multisubunit glycoproteins are recognized by the endoplasmic reticulumassociated degradation (ERAD) pathway regulated largely by their N-linked polymannose oligosaccharides. In this quality control system, lectin-like molecular chaperones, calnexin and calreticulin, recognize the Glc 1 Man 9 GlcNAc 2 oligosaccharide and assist in folding of newly synthesized glycoproteins. Lectin interaction with Glc 1 Man 9 GlcNAc 2 glycans after trimming with ER alpha-glucosidases, and alpha-mannosidases sorts out persistently unfolded glycoproteins for N-deglycosylation and degradation by the proteasome (Cresswell and Hughes, 1997;Kopito, 1997;Brodsky and McCracken, 1999;Romisch, 1999;Spiro, 2004).The mannose trimming of N-linked glycans plays an important role in the ERAD of glycoproteins (Spiro, 2004). In both yeast and human cells, it is reported that the misfolded glycoproteins in the ER are degraded through ERAD only after the glycan is trimmed to the Man8B form, while misfolded proteins stay within the ER when they retain the Man9 form of oligosaccharides. Accordingly, it was assumed that there existed a Man8-binding lectin, later identified as EDEM, that recognizes misfolded glycoproteins in the Man8B form in the ER and directs them to the ERAD pathway (Hosokawa et al., 2001). The misfolded glycoproteins exit the ER via a multiprotein complex referred to as a retrotranslocon or dislocon (Plemper et al., 1997;Bebok et al., 1998;de Virgilio et al., 1998;Tsai et al., 2002). In alternative models both PNGase and proteasomes may be either free in the cytosol or ER membrane imbedded or attached (Spiro, 2004).A well-studied model for the ERAD pathway utilizes the human cytomegalovirus, which affects the MHC class I antigen presentation. The vira...

Peptide:N-glycanase (PNGase) is a deglycosylating enzyme that catalyzes the hydrolysis of the b-aspartylglycosylamine bond of aspargine-linked glycopeptides and glycoproteins. Earlier studies from our laboratory indicated that PNGase catalyzed de-N-glycosylation was limited to glycopeptide substrates, but recent reports have demonstrated that it also acts upon full-length misfolded glycoproteins. In this study, we utilized two glycoprotein substrates, yeast carboxypeptidase and chicken egg albumin (ovalbumin), to study the deglycosylation activity of yeast PNGase and its mutants. Our results provide further evidence that PNGase acts upon full-length glycoprotein substrates and clearly establish that PNGase acts only on misfolded or denatured glycoproteins.

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