Editors' ChoiceCell Biology

Degron for autophagic disposal

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Science Signaling  21 Jul 2015:
Vol. 8, Issue 386, pp. ec198
DOI: 10.1126/scisignal.aad0438

Proteolysis of proteins that results in the exposure of a destabilizing N-terminal residue in a specific amino-acid sequence (an N-degron) can result in targeting of the protein for degradation by the ubiquitin (Ub)–proteasome system (see Martens and Bachmair). Yu et al. found an N-degron involving N-terminal arginylation by arginyltransferase 1 (ATE1) that enables degradation of targeted proteins by autophagy. Unexpectedly, bioinformatic analysis revealed that many proteins targeted to the endoplasmic reticulum (ER), including the chaperones BiP and CRT and the protein disulfide isomerase PDI, were potential substrates for arginylation. An antibody specific for the arginylated form of BiP (R-BiP) did not detect R-BiP in unstressed cultured cells, but R-BiP accumulated in the cytosol in cells overexpressing ATE1 or in cells transfected with various sources of double-stranded DNA (dsDNA), including poly(dA:dT). Although detectable in unstressed cells, the abundance of R-BiP increased in cells transfected with dsDNA. Immunofluorescence analysis indicated that poly(dA:dT) stimulated the colocalization of R-BiP with the autophagosomal proteins p62 and LC3 in a manner dependent on ATE1. R-BiP lacking the ATPase and substrate-binding domain (R-BiPΔ) also colocalized with p62 and LC3, and pull-down assays with p62 mutants indicated that the ZZ domain of p62 recognized R-BiP. The dipeptide Arg-Ala, but not other dipeptides, stimulated the oligomerization and aggregation of p62 in extracts of cells overexpressing p62 and increased the interaction of the overexpressed p62 in the cell extracts with immobilized LC3. When expressed in cells, glutathione-S-transferase (GST) fused to R-BiPΔ was less abundant than GST fused to a nonarginylated mutant V-BiPΔ, indicating that R-BiP functioned as a degron, and the amount of R-BiPΔ-GST was greater when autophagy was inhibited. Cells exposed to poly(dA:dT) exhibited an increase in ubiquitylated proteins as well as an increase in the abundance of R-BiP, R-CRT, and R-PDI and Ub-positive puncta colocalized with p62 and R-BiP, suggesting that R-BiP may interact with the misfolded proteins to deliver them for autophagic clearance.

H. Cha-Molstad, K. S. Sung, J. Hwang, K. A. Kim, J. E. Yu, Y. D. Yoo, J. M. Jang, D. H. Han, M. Molstad, J. G. Kim, Y. J. Lee, A. Zakrzewska, S.-H. Kim, S. T. Kim, S. Y. Kim, H. G. Lee, N. K. Soung, J. S. Ahn, A. Ciechanover, B. Y. Kim, Y. T. Kwon, Amino-terminal arginylation targets endoplasmic reticulum chaperone BiP for autophagy through p62 binding. Nat. Cell Biol. 17, 917–929 (2015). [PubMed]

S. Martens, A. Bachmair, How cells coordinate waste removal through their major proteolytic pathways. Nat. Cell Biol. 17, 841–842 (2015). [PubMed]