The Retrotranslocation Protein Derlin-1 Binds Peptide:N-Glycanase to the Endoplasmic Reticulum

Abstract

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...

The Role of a Novel p97/Valosin-containing Protein-interacting Motif of gp78 in Endoplasmic Reticulum-associated Degradation

“…In addition, other cytosolic ERAD components are also recruited to the ER by the multiprotein complex. For examples, both Derlin-1 and p97/ VCP interact with the peptide:N-glycanase, a key enzyme that removes N-glycan from misfolded ER proteins before proteasomal degradation (39,40). Peptide:N-glycanase also interacts with hHR23B, a polyubiquitin-binding protein that chaperones polyubiquitinated ERAD substrates to the proteasomes for degradation (39,41).…”

Selenoprotein K Binds Multiprotein Complexes and Is Involved in the Regulation of Endoplasmic Reticulum Homeostasis

“…Inclusion of a proteasome inhibitor in the experiment led to an accumulation of the deglycosylated forms of substrates (Fig. 5, D and E, compare lanes 7 and 5), whose N-linked glycans are removed by a cytosolic N-glycanase attached to Derlin-1 (17). Knockdown of SelK in MG132-treated cells did not affect clearance of the HA-tagged NHK (Fig.…”

“…First, these proteins are involved in or are required for degradation of many ERAD substrates (8 -14). Second, Derlin-1 and -2 were found to interact with lumenal (8,11), membrane (15,16) and cytosolic (9,14,17) proteins required for retrotranslocation. Finally, Derlins are capable of forming homo-and heterooligomers (11,15,16,18), which is consistent with their ability to form large complexes and serve as channels.…”

Generation and Metabolism of Cytosolic Free Oligosaccharides in Caenorhabditis elegans

“…Further- more, mammalian PNGase forms protein complexes via its N-terminal peptide:N-glycanase/UBA or UBX-containing protein (PUB) domain with other molecules playing central roles in ERAD such as p97/Cdc48/VCP (cytosolic AAA ATPase), Derlin-1 (putative retrotranslocation channel), and AMFR (ER-associated ubiquitin ligase E3). This demonstrates that PNGase is profoundly involved in ERAD as a deglycosylating enzyme for substrate proteins (21,22).…”

“…The generation of FOSs was directly linked to ERAD because the cytoplasmic product of the PNGase gene ( png1) from yeast S. cerevisiae is known to interact with the 20S proteasome. Many subsequent reports of ERAD-related protein interactions have revealed that PNGase, a FOSproducing enzyme, is a major component of the ERAD pathway (19)(20)(21)(22).…”