The N-End Rule Pathway

Supplementary Data

• Download Supplemental Figures 1-6 (PDF), or see below.

  Supplemental Figure 1. A model of the regulation of cardiovascular signaling through controlled proteolysis of G protein regulators by the N-end rule pathway.

  

  The degradation of RGS4, RGS5, and RGS16 is initiated by cotranslational cleavage of N-terminal Met by MetAPs, which exposes the pro-N-degron Cys2 at the N terminus. In normally growing cells, N-terminally exposed Cys2 undergoes a cotranslational redox modification into sulfinic acid [CysO₂(H)] or Cys sulfonic acid [CysO₃(H)]; this modification is inhibited by depletion of oxygen or nitric oxide. The resulting oxidized Cys residue structurally mimics the secondary destabilizing residue Asp and thus can serve as an acceptor substrate for ATE1 R-transferases. Following arginylation, these RGS proteins are targeted by N-recognins for proteasomal degradation, resulting in the activation of PGCR signaling. In this model, RGS proteins with Cys2 are constitutively degraded to turn up G protein signaling in hearts and blood vessels under normal physiological conditions in which cells are exposed to sufficient O₂ and NO; this allows cells to sense extracellular ligands at a maximal level. However, when O₂ (or other molecules that induce the oxidation of Cys2) in circulating blood is not sufficient, for example, in ischemia caused by cardiac arrest or other cellular stresses, these substrates are rapidly accumulated in a real-time basis to turn down GPCR signaling, decoupling cells from extracellular proliferation signals. Thus, arginylation-induced proteolysis may function as a cellular stress response to maintain homeostasis in GPCR signaling in the heart (via RGS4) and blood vessels (via RGS5). Pa, a palmitoyl group conjugated to the N-terminal Cys residue.

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  Supplemental Figure 2. The regulation of apoptosis by the Drosophila N-end rule pathway.

  

  (a) The degradation of DIAP1 by the N-end rule pathway. When the effector caspase drICE cleaves off the N-terminal 20 residues of DIAP1, the pro-N-degron Asn-21 is exposed at the N terminus of the resulting C-terminal DIAP1 fragment. Asn-21 undergoes sequential N-terminal modifications by Drosophila (dr)-NTAN1 (deamidation) and dr-ATE1 (arginylation). The arginylated N terminus of the DIAP1 fragment is recognized by Drosophila UBR E3s for ubiquitylation and proteasomal degradation. Both the full-length DIAP1 and the N-terminally cleaved DIAP1 can be degraded though its autoubiquitylation activity.
  
  (b) A UBR E3 and DIAP1 may form a heterodimeric E3 complex. The arginylated DIAP1 fragment cooperates with a UBR box protein, such as drUBR1, to inhibit drICE caspase activity through nondegradative polyubiquitylation. In this model, the DIAP1 fragment is not the N-end rule substrate but is linked to drUBR1 through the UBR box (red), while the BIR domain (orange) recognizes the IAP-binding motif (IBM) of drICE. Each RING domain (yellow) recruits a cognate E2 enzyme. A cognate E2 for drUBR1 may be UbcD6, a homolog of yeast Rad6.

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  Supplemental Figure 3. Proteolytic mechanisms of the N-end rule pathways in eukaryotes and prokaryotes.

  

  (a) The ClpS-ClpAP proteolytic machinery in bacteria. In the bacterial N-end rule pathway, the N-recognin ClpS binds to the destabilizing N-terminal residue (small blue circle) of a substrate (green oval). The ClpS-bound substrate is directly delivered to the ClpAP complex, a ring-shaped proteolytic machinery, which functions like the 26S proteasome.
  
  (b) The ubiquitin-proteasome proteolytic machinery in eukaryotes. In the eukaryotic N-end rule pathway, an N-recognin, in conjunction with E2, binds to the N-terminal destabilizing residue of a substrate and mediates the conjugation of ubiquitin to the Lys residue of the substrate. Ubiquitin functions as a secondary degradation signal for efficient delivery into the proteolytic chamber of the 26S proteasome.

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  Supplemental Figure 4. The dual E1-E2 system in N-end rule pathway.

  

  Canonical UBR proteins (UBR1, UBR2, and UBR3) mediate ubiquitylation in conjunction with either of two distinctive E1-E2 ubiquitin activation/conjugation systems, UBA6-USE1 or UBA1-RAD6. USE1, a UBA6-specific E2, exclusively accepts the activated ubiquitin from the E1 enzyme UBA6. Both of the E2 enzymes RAD6 and USE1 bind these E3s through the RING domain. UBA6 is enriched in the cytoplasm, whereas UBA1 is localized in both the cytoplasm and the nucleus. The N-end rule substrates RGS4 and RGS5 are mediated by UBR2 through the dual E1-E2 system. "∼S∼" denotes a thioester bond between the last residue (Gly76) of ubiquitin (small orange oval) and an active Cys of E1 or E2. The oval with "S" denotes a substrate. "Ub(n)" denotes a polyubiquitin chain.

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  Supplemental Figure 5. The regulation of the peptide import by the S. cerevisiae N-end rule pathway.

  

  (a) A schematic model of the Cup9 degradation and the autoinhibitory feedback. S. cerevisiae Cup9 is depicted by a light blue oval. Dipeptides bearing the destabilizing residues are depicted by a small red oval for type 1 and by a blue triangle for type 2. Yeast Ubr1 recognizes an internal degron (I-degron in red) in the C-terminal region of Cup9 through a protein-protein interaction. The Cup9 binding site (pale green) of Ubr1 is localized within the N-terminal half (∼100 kDa) of Ubr1 and does not apparently overlap with either UBR box or N domain. The availability of the Cup9 binding site is regulated by the autoinhibitory (AI) domain (bright green) and the concentration of type-1/2 dipeptides, whereas the E2 Rad6 binding site is constitutively available. Different lengths of polyubiquitin chains are depicted to indicate the processivity of polyubiquitylation by Ubr1-E2, which reflects the availability of dipeptides. Brackets indicate the concentration of Cup9. The diagram (bottom) shows the feedback loop of the Cup9 degradation by Ubr1.
  
  (b) The phosphorylation of Ser300 on the yeast Ubr1 by the casein kinases Yck1/2 in the response to extracellular amino acids is important for the degradation of Cup9. The Ser300 is located between the UBR box and N domain, indicating that the Ser300 may be involved in the autoinhibitory mechanism.

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  Supplemental Figure 6. Strategies to use engineered N-degrons as a molecular tool.

  

  (a) The ubiquitin-fusion technique. In eukaryotes, ubiquitin-protein fusions can be cotranslationally cleaved by deubiquitinating enzymes (DUBs) at the C-terminal residue of Ub, generating a protein of interest (POI) with a destabilizing residue.
  
  (b) A portable N-degron can be fused with the POI to induce proteasomal degradation.
  
  (c) A temperature-sensitive N-degron (td) can induce conditional degradation through N-end rule pathway. At 23°C, the ts DHFR mutant is stable even though it has an N-terminal Arg. By increasing the temperature to 37°C, the ts Arg-DHFR mutant undergoes a conformational change, making the N-terminal region accessible to N-recognin. This conformational change is reversible so that the stability of td-fused protein is controlled by temperature switching.