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. 2006 May 30;103(22):8348-53.
doi: 10.1073/pnas.0602747103. Epub 2006 May 18.

The AAA ATPase p97 links peptide N-glycanase to the endoplasmic reticulum-associated E3 ligase autocrine motility factor receptor

Guangtao Li  1 , Gang Zhao, Xiaoke Zhou, Hermann Schindelin, William J Lennarz
Affiliations

The AAA ATPase p97 links peptide N-glycanase to the endoplasmic reticulum-associated E3 ligase autocrine motility factor receptor

Guangtao Li et al. Proc Natl Acad Sci U S A. .

Abstract

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.

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Conflict of interest statement

Conflict of interest statement: No conflicts declared.

Figures

Fig. 1.
Fig. 1.
Colocalization of mAMFR-myc and GFP-mPNGase. COS1 cells transiently expressing GFP-mPNGase (green) and mAMFR-myc (red) were assessed for colocalization by confocal microscopy after staining with anti-myc Ab. The merged panel shows colocalization (yellow) of mAMFR and GFP-mPNGase in the ER-enriched perinuclear region of the cell.
Fig. 2.
Fig. 2.
GFP-mPNGase was coimmunoprecipitated with mAMFR-myc in COS1 cells. COS1 cells were cotransfected with plasmids encoding GFP and mAMFR-myc (lanes 2 and 4) and GFP-mPNGase and mAMFR-myc (lanes 1 and 3). Cell lysates were subject to immunoprecipitation (IP) with monoclonal anti-Myc Ab (lanes 3 and 4) in RIPA buffer. The monoclonal anti-myc Ab and bound proteins were recovered with agarose beads containing coupled protein A. The cell lysate (lanes 1 and 2) and the proteins coupled to the protein G beads (lanes 3 and 4) were subjected to SDS/PAGE and immunoblotted (IB) with polyclonal anti-GFP (Upper) or anti-myc (Lower).
Fig. 3.
Fig. 3.
The PUB domain of mPNGase is responsible for the interaction with His6-mp97. (A) mPNGase constructs and the results of interactions between the indicated mPNGase variants and His6-mp97. (B) The PUB domain of mPNGase is responsible for the interaction with His6-mp97. GST fusion protein containing mPNGase variants was immobilized on glutathione beads and incubated with purified His6-mp97. Bound proteins were subjected to SDS/PAGE and blotted with monoclonal anti-GST (Upper) or anti-His Ab (Lower). The asterisk in lane 4 indicates degraded GST-mPNGase (1–181). (C) Sequence alignments, with conserved amino acids in bold. Amino acids chosen for mutation are labeled with a diamond. M. mus., Mus musculus; D.mel., Drosophila melanogaster; A. tha., Arabidopsis thalania. (D) The N41P and G79A/F80A mutants abolish the interaction between mPNGase and mp97. Pull-down experiments were as described in B.
Fig. 4.
Fig. 4.
Different regions on mp97 mediate binding to mPNGase, mAMFR, and mY33K. (A) Schematic diagram of the different regions of mp97. GST-mPNGase (1–181) (B) or GST-mY33K (C) bound on glutathione beads was incubated with His6-tagged mp97 variants. Bound proteins were separated by SDS/PAGE and blotted with monoclonal anti-His Ab. (B) The C terminus of mp97 associates with mPNGase. (C) The N terminus of mp97 interacts with mY33K.
Fig. 5.
Fig. 5.
mp97 mediates formation of three ternary complexes, mAMFR–mp97–mPNGase, mPNGase–mp97–mY33K, and mAMFR–mp97–mY33K and a quintenary complex containing mAMFR, mp97, mY33K, mPNGase, and mHR23B. (A) Formation of the ternary mAMFR(c)–mp97–mPNGase complex. GST-mAMFR(c) (0.25 μM) bound on glutathione beads was incubated with 0.25 μM His6-mp97 in the presence of a 0- to 10-fold molar excess of His6-mPNGase. Bound proteins were subjected to SDS/PAGE and blotted with anti-mPNGase (Top), anti-mp97 (Middle), or anti-GST (Bottom) Ab. (B) Formation of the ternary of mPNGase–mp97–mY33K complex. GSH-agarose beads containing 0.25 μM GST-mPNGase(1–181) were incubated with 0.25 μM His6-mp97 and then incubated with or without 0.5 μM His6-mY33K. (C) Formation of the ternary mAMFR–mp97–mY33K complex. Chitin beads containing 0.25 μM intein-mAMFRc49 were incubated with 0.25 μM His6-mp97 and then incubated with 0, 0.25, or 2.5 μM His6-mY33K. Bound proteins were subjected to SDS/PAGE and blotted with a monoclonal anti-His Ab (C and D). (D) Formation of a quintenary complex. Chitin beads containing 0.25 μM intein-mAMFRc49 were incubated with 2.5 μM His6-mp97 and then harvested and successively incubated with 2.5 μM His6-mY33K, His6-mPNGase, and His6-mHR23B. The asterisk in lane 5 indicates His6-mPNGase and degraded His6-mp97.
Fig. 6.
Fig. 6.
Model depicting the events of retrotranslocation, ubiquitination, deglycosylation, and degradation of a glycosylated ERAD substrate. Direct interactions among the five proteins mAMFR, mp97, mY33K, mPNGase, and mHR23B are depicted as determined in this study. Interactions among Derlin-1, p97, mAMFR (7, 8), mHR23B, and proteasome were reported in ref. . The arrangement of the glycoprotein being degraded is hypothetical.
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