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

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 aKdof 16 μM, whereas the otherwise identical Nt-Pro–bearing sequence PGLW binds to GID4 more tightly, with aKdof 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 yeastSaccharomyces cerevisiaecan 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.

Timer-based proteomic profiling of the ubiquitin-proteasome system reveals a substrate receptor of the GID ubiquitin ligase

“…The vertebrate GID/CTLH (C-terminal to LisH) complex is linked to a variety of processes, including cilium function, the cell cycle, and metabolism ( Boldt et al., 2016 ; Lampert et al., 2018 ; Leal-Esteban et al., 2018 ; Liu and Pfirrmann, 2019 ; Liu et al., 2020 ; Pfirrmann et al., 2015 ; Texier et al., 2014 ). Yeast and human Gid4 appear to recognize similar N-degrons, although no substrates of human Gid4 are known ( Chen et al., 2017 ; Dong et al., 2018 , 2020 ), and N termini of known substrates do not fit the Gid4 consensus ( Lampert et al., 2018 ). It will therefore be important to understand how the expanded specificity of the GID complex described here translates outside of yeast.…”

GID E3 ligase supramolecular chelate assembly configures multipronged ubiquitin targeting of an oligomeric metabolic enzyme

“…We tested if the structural conservation extended to enzymatic mechanism. Because Pro/Nend degron targets of the CTLH E3 remain unknown, we generated a model peptide substrate: an N-terminal PGLW sequence, previously reported to optimally bind hGid4 (Dong et al, 2020;Dong et al, 2018), connected via a flexible linker to a C-terminal target lysine.…”

“…The CTLH E3, named based on the preponderance of CTLH domains (in Gid1, Gid2, Gid7, Gid8 and Gid9 and their orthologs across evolution), has intrinsic E3 ligase activity (Lampert et al, 2018;Liu and Pfirrmann, 2019;Maitland et al, 2019). Although Pro/N-degron substrates of the CTLH E3 are yet to be identified, human Gid4 structurally superimposes with yeast Gid4 assembled into GID SR4 and likewise preferentially binds peptides with an N-terminal proline (Dong et al, 2020;Dong et al, 2018;Qiao et al, 2020).…”

The human GID complex engages two independent modules for substrate recruitment

“…Cross-linking Mass Spectroscopy: All cross-link identifications are provided in SI Tables 1, 2, 4 ubiquitin and the 6-subunit hGID complex (ARCM8, RanBP9, WDR26, MAEA, RMND5a, and TWA1) in the presence or absence of GID4 and a 10-fold excess of the PGLV GID4-specific peptide. F. Comparison of the N-terminal sequences of the first five amino acids of the Pro-/N-degron consensus motif (Dong et. al.…”

“…In addition to Gid4, yeast cells express two alternative substrate receptors, Gid10 and Gid11, which all interact with the GID E3 ligase complex through Gid5/ARMC8 (Melnykov et al, 2019, Edwin Kong et al, 2021. Gid4 and Gid10 bind substrates containing an N-Pro degron motif (Dong et al, 2020), although systematic screening identified many candidates that do not fulfill these criteria (Edwin Kong et al, 2021). Similarly, human GID4 may also recognize substrates like ZMYND19 that lack N-Pro degron motifs.…”