Recognition of nonproline N-terminal residues by the Pro/N-degron pathway

Cheng Dong; Shun-Jia Chen; Artem Melnykov; Sara Weirich; Kelly Sun; Albert Jeltsch; Alexander Varshavsky; Jinrong Min

doi:10.1073/pnas.2007085117

New Research In

Edited by F. Ulrich Hartl, Max Planck Institute of Chemistry, Martinsried, Germany, and approved May 13, 2020 (received for review April 14, 2020)

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Significance

The Pro/N-degron pathway targets proteins for degradation through their N-terminal (Nt) proline (Pro) residue. GID4, a subunit of the GID ubiquitin ligase, is the recognition component of the Pro/N-degron pathway. Here we show that human GID4 can also recognize hydrophobic Nt-residues other than Pro. In agreement with these results, a protein bearing the Nt-sequence IGLW was found to be targeted for degradation by the yeast Pro/N-degron pathway. We solved crystal structures of human GID4 bound to specific peptides, and explored the roles of GID4 residues in the targeting of its substrates. These and related results advance the understanding of the Pro/N-degron pathway and expand the substrate recognition range of the GID ubiquitin ligase in both human and yeast cells.

Abstract

Eukaryotic N-degron pathways are proteolytic systems whose unifying feature is their ability to recognize proteins containing N-terminal (Nt) degradation signals called N-degrons, and to target these proteins for degradation by the 26S proteasome or autophagy. GID4, a subunit of the GID ubiquitin ligase, is the main recognition component of the proline (Pro)/N-degron pathway. GID4 targets proteins through their Nt-Pro residue or a Pro at position 2, in the presence of specific downstream sequence motifs. Here we show that human GID4 can also recognize hydrophobic Nt-residues other than Pro. One example is the sequence Nt-IGLW, bearing Nt-Ile. Nt-IGLW binds to wild-type human GID4 with a K_d of 16 μM, whereas the otherwise identical Nt-Pro–bearing sequence PGLW binds to GID4 more tightly, with a K_d of 1.9 μM. Despite this difference in affinities of GID4 for Nt-IGLW vs. Nt-PGLW, we found that the GID4-mediated Pro/N-degron pathway of the yeast Saccharomyces cerevisiae can target an Nt-IGLW–bearing protein for rapid degradation. We solved crystal structures of human GID4 bound to a peptide bearing Nt-Ile or Nt-Val. We also altered specific residues of human GID4 and measured the affinities of resulting mutant GID4s for Nt-IGLW and Nt-PGLW, thereby determining relative contributions of specific GID4 residues to the GID4-mediated recognition of Nt-Pro vs. Nt-residues other than Pro. These and related results advance the understanding of targeting by the Pro/N-degron pathway and greatly expand the substrate recognition range of the GID ubiquitin ligase in both human and yeast cells.

Footnotes

↵¹C.D. and S.-J.C. contributed equally to this work.
↵²To whom correspondence may be addressed. Email: avarsh{at}caltech.edu or jr.min{at}utoronto.ca.

Author contributions: C.D., S.-J.C., A.M., A.J., A.V., and J.M. designed research; C.D., S.-J.C., A.M., S.W., and K.S. performed research; C.D., S.-J.C., A.M., S.W., K.S., A.J., A.V., and J.M. analyzed data; and C.D., S.-J.C., A.M., A.J., A.V., and J.M. wrote the paper.
The authors declare no competing interest.
This article is a PNAS Direct Submission.
Data deposition: The atomic coordinates and structure factors of GID4-IGLWKS and GID4-VGLWKS complexes have been deposited in the Protein Data Bank (https://www.rcsb.org/) under the accession codes 6WZX and 6WZZ, respectively.
This article contains supporting information online at https://www.pnas.org/lookup/suppl/doi:10.1073/pnas.2007085117/-/DCSupplemental.

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