During endoplasmic reticulum-associated degradation, the multifunctional AAA ATPase p97 is part of a protein degradation complex. p97 associates via its N-terminal domain with various cofactors to recruit ubiquitinated substrates. It also interacts with alternative substrate-processing cofactors, such as Ufd2, Ufd3, and peptide:N-glycanase (PNGase) in higher eukaryotes. These cofactors determine different fates of the substrates and they all bind outside of the N-terminal domain of p97. Here, we describe a cofactor-binding motif of p97 contained within the last 10 amino acid residues of the C terminus, which is both necessary and sufficient to mediate interactions of p97 with PNGase and Ufd3. The crystal structure of the N-terminal domain of PNGase in complex with this motif provides detailed insight into the interaction between p97 and its substrate-processing cofactors. Phosphorylation of p97's highly conserved penultimate tyrosine residue, which is the main phosphorylation site during T cell receptor stimulation, completely blocks binding of either PNGase or Ufd3 to p97. This observation suggests that phosphorylation of this residue modulates endoplasmic reticulum-associated protein degradation activity by discharging substrate-processing cofactors.PUB domain ͉ Ufd3 ͉ endoplasmic reticulum-associated protein degradation E ndoplasmic reticulum-associated protein degradation (ERAD) is a major component of the quality control system in the protein secretory pathway. It is responsible for the removal of misfolded proteins and unassembled protein subunits (1, 2). Once (glyco-)proteins are recognized for degradation in the endoplasmic reticulum, they are routed to the cytosol via the putative retrotranslocon, polyubiquitinated, and degraded by the ubiquitin-proteasome system (3). It has been proposed that ERAD substrates are threaded mechanically through the retrotranslocon by the chaperone-like protein p97 (CDC48 in yeast) (4), which is also known as VCP (valosin-containing protein). p97 belongs to the AAA ATPase family of proteins (5), and its sequence is highly conserved in eukaryotes. RNAi and mutagenesis experiments have confirmed the essential role of p97 in the proteasome-mediated degradation of misfolded proteins (6). Structural studies (7) revealed that p97 has an N-terminal domain (N), two AAA ATPase domains (D1 and D2), and a short disordered C-terminal region (C). In addition to its role in ERAD, p97 has been found to participate in other cellular processes, including DNA repair, cell cycle control, and membrane trafficking (4, 8). Structural and biochemical studies have suggested that p97 fulfills its various functions by interacting with a wide spectrum of cofactors (9-12).Most of its cofactors interact with the N-terminal domain of p97, with the exceptions of Ufd2, Ufd3, and peptide:N-glycanase (PNGase), which interact with the C-terminal region of p97 (9, 12, 13). The cofactors can be divided into substrate-recruiting cofactors, which regulate the substrate specificity of p97, and substrate-processi...
Mouse peptide N-glycanase (mPNGase) cleaves the N-glycan chain from misfolded glycoproteins and glycopeptides. Previously, several proteins were found to directly interact with mPNGase; among them, both mHR23B and mS4 were found to link mPNGase to the proteasome. In this study, we found that the cytoplasmic protein mp97 participates in the formation of a ternary complex containing mouse autocrine motility factor receptor (mAMFR), mp97, and mPNGase. This assemblage recruits the cytosolic mPNGase close to the endoplasmic reticulum (ER) membrane, where the retrotranslocation of misfolded glycoproteins is thought to occur. In addition to the ER membrane-associated E3 ligase mAMFR, a cytosolic protein mY33K, containing both UBA and UBX domains, was found to also directly interact with mp97. Thus, a complex containing five proteins, mAMFR, mY33K, mp97, mPNGase, and mHR23B, is formed in close proximity to the ER membrane and serves to couple the activities of retrotranslocation, ubiquitination, and deglycosylation and, thereby, route misfolded glycoproteins to the proteasome.endoplasmic reticulum-associated degradation ͉ retrotranslocation ͉ proteasome ͉ ubiquitination ͉ deglycosylation
pnas.0509057102), the numerical scales corresponding to the color bars in Figs. 2, 4, and 5 appeared incorrectly, due to a printer's error. The corrected figures and their legends appear below.
Peptide:N-glycanase (PNGase) is an important component of the endoplasmic reticulum-associated protein degradation pathway in which it de-glycosylates misfolded glycoproteins, thus facilitating their proteasomal degradation. PNGase belongs to the transglutaminase superfamily and features a Cys, His, and Asp catalytic triad, which is essential for its enzymatic activity. An elongated substrate-binding groove centered on the active site Cys191 was visualized in the crystal structure of apo-PNGase, whereas its complex with Z-VAD-fmk, a peptide-based inhibitor of PNGase, revealed that the inhibitor occupied one end of the substrate-binding groove while being covalently linked to the active site Cys. Recently, haloacetamidyl-containing carbohydrate-based inhibitors of PNGase were developed and shown to specifically label the active site Cys. In this study, we describe the crystal structure of yeast PNGase in complex with N,N'-diacetylchitobiose (chitobiose). We found that the chitobiose binds on the side opposite to the peptide binding site with the active site Cys191 being located approximately midway between the carbohydrate and peptide binding sites. Mutagenesis studies confirm the critical role of the chitobiose-interacting residues in substrate binding and suggest that efficient oligosaccharide binding is required for PNGase activity. In addition, the N-terminus of a symmetry-related PNGase was found to bind to the proposed peptide-binding site of PNGase. Together with the bound chitobiose, this enables us to propose a model for glycoprotein binding to PNGase. Finally, deleting the C-terminal residues of yeast PNGase, which are disordered in all structures of this enzyme, results in a significant reduction in enzyme activity, indicating that these residues might be involved in binding of the mannose residues of the glycan chain.
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