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Amino-terminal arginylation targets endoplasmic reticulum chaperone BiP for autophagy through p62 binding

Nature Cell Biology volume 17, pages 917–929 (2015)Cite this article

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Abstract

We show that ATE1-encoded Arg-transfer RNA transferase (R-transferase) of the N-end rule pathway mediates N-terminal arginylation of multiple endoplasmic reticulum (ER)-residing chaperones, leading to their cytosolic relocalization and turnover. N-terminal arginylation of BiP (also known as GRP78), protein disulphide isomerase and calreticulin is co-induced with autophagy during innate immune responses to cytosolic foreign DNA or proteasomal inhibition, associated with increased ubiquitylation. Arginylated BiP (R-BiP) is induced by and associated with cytosolic misfolded proteins destined for p62 (also known as sequestosome 1, SQSTM1) bodies. R-BiP binds the autophagic adaptor p62 through the interaction of its N-terminal arginine with the p62 ZZ domain. This allosterically induces self-oligomerization and aggregation of p62 and increases p62 interaction with LC3, leading to p62 targeting to autophagosomes and selective lysosomal co-degradation of R-BiP and p62 together with associated cargoes. In this autophagic mechanism, Nt-arginine functions as a delivery determinant, a degron and an activating ligand. Bioinformatics analysis predicts that many ER residents use arginylation to regulate non-ER processes.

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Figure 1: Bioinformatic analysis of the ER N-end rule pathway, and the generation of antibodies to the arginylated form of BiP.
Figure 2: ATE1-dependent N-terminal arginylation of multiple ER-residing proteins is induced by cytosolic foreign dsDNA.
Figure 3: R-BiP is targeted to the autophagosome by way of p62 bodies.
Figure 4: The Nt-arginine residue of R-BiP is a delivery determinant to the autophagosome.
Figure 5: The Nt-arginine residue of R-BiP binds to the ZZ domain of p62.
Figure 6: The Nt-arginine residue induces oligomerization and aggregation of p62 in vitro.
Figure 7: R-BiP is induced by and associated with cytosolic misfolded proteins, and ATE1-deficient cells are hypersensitive to misregulation of protein quality control.
Figure 8: A model illustrating the role of the N-end rule pathway in N-terminal arginylation of ER-residing proteins and the ligand-mediated regulation of autophagy in stressed cells.

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Acknowledgements

We thank S. K. Ko (KRIBB) for providing HeLa cells stably expressing RFP–GFP–LC3, Suhyun Lee (Seoul National University) for immunoblotting analysis of R-BiP, W. T. Kwon (Columbia University) for bioinformatics analysis of N-degrons in the ER, H. J. Jeong (KAIST) for immunostaining analysis of R-BiP and KDEL, S. Hong (Yonsei University) for immunostaining analysis of NFκB, and S. J. Yoo (Middleton High School) for technical assistance. This work was supported by the World Class Institute (WCI) Program (WCI 2009-002 to B.Y.K.) and the Bio and Medical Technology Development Program (NRF-2014M3A9B5073938 to B.Y.K.) of the National Research Foundation (NRF) funded by the Ministry of Science, ICT and Future Planning (MSIP) of Korea, the Global R&D Center (GRDC) Program (to J.S.A.), the KRIBB Research Initiative Program, NIH grant HL083365 (to Y.T.K. and S. Li), the Basic Science Research Programs of the NRF funded by the MSIP (NRF-2013R1A2A2A01014170 to Y.T.K.) and by the Ministry of Education (NRF-2013R1A1A2058983 to Y.D.Y.), the Brain Korea 21 PLUS Program (to SNU), the SNU Nobel Laureates Invitation Program (to A.C.), the Miriam and Sheldon G. Adelson Medical Research Foundation (AMRF) (to A.C.) and the Israel Science Foundation (ISF) (to A.C.). A.C. is an Israel Cancer Research Fund (ICRF) USA Professor.

Author information

Author notes

  1. Jun Min Jang

    Present address: Present address: Quality Control Division, Yuhan Chemical, Ansan, Kyung Kee 425-836, Korea.,

  2. Hyunjoo Cha-Molstad, Ki Sa Sung, Joonsung Hwang and Kyoung A. Kim: These authors contributed equally to this work.

Authors and Affiliations

  1. World Class Institute, Korea Research Institute of Bioscience and Biotechnology, Ochang 363-883, Cheongwon, Korea

    Hyunjoo Cha-Molstad, Joonsung Hwang, Kyoung A. Kim, Ji Eun Yu, Jung Gi Kim, Su-Hyeon Kim, Nak Kyun Soung & Bo Yeon Kim

  2. Protein Metabolism Medical Research Center and Department of Biomedical Sciences, College of Medicine, Seoul National University, Seoul 110-799, Korea

    Ki Sa Sung, Young Dong Yoo, Michael Molstad, Yoon Jee Lee, Sung Tae Kim, Aaron Ciechanover & Yong Tae Kwon

  3. Center for Pharmacogenetics and Department of Pharmaceutical Sciences, School of Pharmacy, University of Pittsburgh, Pittsburgh, Pennsylvania 15261, USA

    Ki Sa Sung, Adriana Zakrzewska & Sung Tae Kim

  4. Department of Drug Discovery and Development, College of Pharmacy, Chungbuk National University, Cheonju 361-736, Chungbuk, Korea

    Ji Eun Yu

  5. Department of Molecular Medicine and Biopharmaceutical Sciences, World Class University (WCU) Program, Seoul National University, Seoul 110-799, Korea

    Jun Min Jang

  6. Department of Applied Chemistry, College of Applied Sciences, Kyung Hee University, Yong-in 446-701, Korea

    Dong Hoon Han

  7. Department of Otolaryngology, Ajou University School of Medicine, Suwon 443-380, Korea

    Sun Yong Kim

  8. Genomic Structure Research Center, Korea Research Institute of Bioscience and Biotechnology, Ochang 363-883, Cheongwon, Korea

    Hee Gu Lee

  9. Chemical Biology Research Center, Korea Research Institute of Bioscience and Biotechnology, Ochang 363-883, Cheongwon, Korea

    Jong Seog Ahn

  10. The Polak Tumor and Vascular Biology Research Center, The Rappaport Faculty of Medicine and Research Institute, Technion-Israel Institute of Technology, Haifa 31096, Israel

    Aaron Ciechanover

  11. Ischemic/Hypoxic Disease Institute, College of Medicine, Seoul National University, Seoul 110-799, Korea

    Yong Tae Kwon

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  1. Hyunjoo Cha-Molstad
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Contributions

Bioinformatic analyses of N-end rule degrons on ER proteins were carried out by M.M.; antibodies to arginylated ER proteins were generated by D.H.H.; immunoblotting analyses of arginylated ER proteins were carried out by K.A.K., Y.D.Y., H.C-M., K.S.S., J.H., J.G.K. and J.E.Y.; immunostaining of arginylated ER proteins was carried out by H.C-M., J.E.Y., Y.J.L. and N.K.S.; DNA-induced innate immune responses were characterized by K.S.S., H.C-M., A.Z., S-H.K., and S.T.K.; the domain of p62 that binds to Nt-arginine was determined by J.M.J. and H.C-M.; the relationship of arginylated ER proteins with misfolded proteins and proteasomal inhibition was investigated by H.C-M. and S.Y.K.; X-peptide pulldown assay with R-BiP peptides was carried out by H.C-M. and J.E.Y. and p62 aggregation assay was carried out by H.C-M. and J.E.Y. H.C-M., K.S.S., J.H. and K.A.K. contributed equally to this work. H.G.L., J.S.A. and B.Y.K. provided guidance, specialized reagents and expertise. Y.T.K., H.C-M., B.Y.K. and A.C. supervised personnel and/or wrote the paper.

Corresponding authors

Correspondence to Bo Yeon Kim or Yong Tae Kwon.

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Integrated supplementary information

Supplementary Figure 5 Sequence alignments of the N-terminal regions of ER-residing chaperone molecules (CRT, PDI, GRP94, and ERdJ5) that acquire evolutionarily conserved Nt-destabilizing residues after the cleavage of their signal peptides.

Red boxes indicate P1′ residues after the cleavage by the signal peptide peptidase.

Supplementary Figure 6 Peptide binding/competition assays using antibodies specific to the arginylation forms of BiP (a), CRT (b) and PDI (c).

(a) An 11-mer R-BiP peptide, which corresponds to the N-terminal region of the arginylated form of BiP, was immobilized on a 96-well plate. A 10-mer peptide (E-BiP peptide) corresponding to an unarginylated form of BiP was used as an N-end rule control. The immobilized peptide was incubated with serially diluted anti-R-BiP antibody, and the amounts of R-BiP antibody bound to immobilized R-BiP peptide were determined using anti-goat secondary antibody conjugated with horseradish peroxide. (b) Similar to a except that the binding of R-CRP and E-CRP peptides to anti-R-CRT antibody was determined. (b) Similar to a except that the binding of R-PDI and E-PDI peptides to anti-R-PDI antibody was determined.

Supplementary Figure 7 R-BiP is present in the cytosol and shows a mutually exclusive localization with the KDEL immunostaining signal which represents the ER.

HEK293 cells (2.5 × 105/well) were incubated in the absence or presence of 200 nM thapsigargin for 6 h, followed by immunostaining of R-BiP in comparison with antibody to the KDEL sequence, the latter representing the ER. Scale bar, 5 μm. (Right) Enlarged views corresponding to the areas indicated by rectangles. Scale bar, 2 μm.

Supplementary Figure 8 Puncta formation and colocalization analysis of R-BiP with LC3 and p62 in HeLa cells treated with poly(dA:dT) dsDNA or 5′-PPP dsRNA.

(a) HeLa cells stably expressing RFP-GFP-LC3 were treated with poly(dA:dT) dsDNA or 5′-PPP dsRNA, followed by immunostaining analysis to determine the formation and colocalization of R-BiP with LC3 puncta as visualized by RFP fluorescence. (b) HeLa cells were treated with poly(dA:dT) dsDNA or 5′-PPP dsRNA, followed by immunostaining analysis to determine the formation and colocalization of R-BiP with p62 puncta.

Supplementary Figure 9 Immunostaining of NF-kB p50 in HeLa cells (3 × 106/well) treated with 0.5 μg/well poly(dA:dT) dsDNA for 16 h.

Upon activation, NF-kB p50 is dissociated from IkB and enters the nucleus to induce the transcription of its target genes, including interferons. Scale bar, 5 μm.

Supplementary Figure 10 Puncta formation and colocalization analysis of p62 in comparison with LC3 which is produced from RFP-GFP-LC3.

HeLa cells expressing RFP-GFP-LC3 were treated with poly(dA:dT) dsDNA, followed by immunofluorescence analysis of acid-resistant RFP-LC3 and acid-sensitive GFP-LC3. Note that most LC3 puncta are positive for both RFP and GFP, indicating that they represent autophagosomes.

Supplementary Figure 11 R-BiP is induced by prolonged proteasomal inhibition and colocalizes with ubiquitin conjugates in autophagic vacuoles.

(a) R-BiP is induced by prolonged proteasomal inhibition. HeLa cells were treated with a proteasomal inhibitor or other stressors, followed by immunoblotting analysis of R-BiP and ubiquitin conjugates (as visualized using FK1 antibody). A23187, calcium ionophore; CCCP, carbonyl cyanide m-chlorophenylhydrazone (Protonophore (H+ ionophore) and uncoupler of oxidative phosphorylation in mitochondria). (b) R-BiP colocalizes with ubiquitin conjugates in autophagic vacuoles. Poly(dA:dT)-treated HeLa cells were subjected to immunostaining analysis of R-BiP and p62 with ubiquitin conjugates as visualized by FK2 antibody. This assay reveals that ubiquitin-positive puncta are invariably positive for both R-BiP and p62. Scale bar, 10 μm. (c) ATE1-knockdown inhibits Nt-arginylation of R-BiP as well as autophagic induction in HeLa cells treated with both 10 μM MG132 and 200 nM thapsigargin. (d) BiP-knockdown inhibits Nt-arginylation of R-BiP as well as autophagic induction in HeLa cells treated with both 10 μM MG132 and 200 nM thapsigargin. (e) R-BiP induced by proteasomal inhibition and ER stress is mainly retrieved from the cytosolic fraction.

Supplementary Table 1 Sequences of the N-terminal regions of ER-residing proteins that carry arginylation-permissive destabilizing N-terminal residues, Asp and Glu.
Full size table
Supplementary Table 2 Sequences of the N-terminal regions of ER-residing proteins that carry tertiary destabilizing N-terminal residues (Asn, Gln, and Cys).
Full size table
Supplementary Table 3 Sequences of the N-terminal regions of ER-residing proteins whose N-terminal Asp and Glu residues are evolutionarily conserved.
Full size table
Supplementary Table 4 Sequences of the N-terminal regions of human ER-residing proteins whose arginylation-permissive residues, Asp and Glu, are not evolutionarily conserved.
Full size table

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Cha-Molstad, H., Sung, K., Hwang, J. et al. Amino-terminal arginylation targets endoplasmic reticulum chaperone BiP for autophagy through p62 binding. Nat Cell Biol 17, 917–929 (2015). https://doi.org/10.1038/ncb3177

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