N-degron pathways

Abstract

An N-degron is a degradation signal whose main determinant is a "destabilizing" N-terminal residue of a protein. Specific N-degrons, discovered in 1986, were the first identified degradation signals in short-lived intracellular proteins. These N-degrons are recognized by a ubiquitin-dependent proteolytic system called the Arg/N-degron pathway. Although bacteria lack the ubiquitin system, they also have N-degron pathways. Studies after 1986 have shown that all 20 amino acids of the genetic code can act, in specific sequence contexts, as destabilizing N-terminal residues. Eukaryotic proteins are targeted for the conditional or constitutive degradation by at least five N-degron systems that differ both functionally and mechanistically: the Arg/N-degron pathway, the Ac/N-degron pathway, the Pro/N-degron pathway, the fMet/N-degron pathway, and the newly named, in this perspective, GASTC/N-degron pathway (GASTC = Gly, Ala, Ser, Thr, Cys). I discuss these systems and the expanded terminology that now encompasses the entire gamut of known N-degron pathways.

Keywords: N-terminal; degron; proteasome; proteolysis; ubiquitin.

Fig. 1.

N-degron pathways in Saccharomyces cerevisiae. (A) The Arg/N-degron pathway. Nt-residues are denoted by single-letter abbreviations. Yellow ovals denote the rest of a protein substrate. “Primary,” “secondary,” and “tertiary” refer to specific classes of destabilizing Nt-residues. “Type 1” and “Type 2” refer to sets of primary destabilizing Nt-residues, basic and bulky hydrophobic. They are recognized by distinct substrate-binding sites of the UBR1 Arg/N-recognin. UBR1 contains other substrate-binding sites as well. 3D structures of the 52-kDa NTA1 Nt-amidase (64), the 58-kDa ATE1 R-transferase (50, 51), and the 225-kDa UBR1 E3 Arg/N-recognin (47) are shown as well (not to scale, owing to a large size of UBR1). Orange (NTA1) and blue (ATE1) denote strongly conserved parts of these enzymes. A multiprotein diagram on the upper right denotes the multienzyme targeting complex of the yeast Arg/N-degron pathway (80). (B) The Ac/N-degron pathway. The red arrowhead on the left indicates the cotranslational removal of Nt-Met by Met-aminopeptidases. If the retained Nt-Met or N-terminal Ala, Ser, or Thr are followed by Nt-acetylation-permissive residues, the above Nt-residues are Nt-acetylated by ribosome-associated Nt-acetylases (53). Nt-Val can be Nt-acetylated but more slowly than, e.g., Nt-Ala, and often negligibly. Nt-Pro is virtually never Nt-acetylated. Nt-Gly is usually Nt-myristoylated, but can also be, alternatively, Nt-acetylated (97). Natural Ac/N-degrons are regulated by their steric shielding in protein complexes (Fig. 5). (C and D) The Pro/N-degron pathway. C and D describe, respectively the initially discovered part of the pathway (67) and the part that involves the aminopeptidases FRA1 and ICP55 (D) (70).

Fig. 2.

(A) The mammalian Arg/N-degron pathway. For designations, see the legend to Fig. 1. This pathway targets proteins for the proteasome-mediated degradation (via the N-recognins UBR1, UBR2, UBR4, UBR5 E3 Ub ligases) or the lysosome-mediated degradation (via the p62 N-recognin). Ub ligases of this pathway can recognize not only the indicated destabilizing Nt-residues but also other degrons in specific protein substrates. NTAN1 and NTAQ1 are Nt-amidases that convert, respectively, Nt-Asn or Nt-Gln to Nt-Asp or Nt-Glu. C* denotes an oxidized N-terminal Cys residue, either Cys-sulfinate or Cys-sulfonate (–45). Five enzymes of the mammalian Arg/N-degron pathway form a targeting complex, analogous to its yeast counterpart (Fig. 1). (B) The mammalian GASTC/N-degron pathway (GASTC = Gly, Ala, Ser, Thr, Cys) (–92), whose name was proposed in this perspective. The CRL-type and IAP-type Ub ligases of the GASTC/N-degron pathway are shown, respectively, above and below the horizontal arrow (, , –92).

Fig. 3.

Activation of NLPR1 sensor by the Arg/N-degron pathway. This diagram of the mouse NLPR1 protein, a sensor of pathogens and other stressors, depicts its domains (NACHT, LRR, ZU5, UPA, CARD) that are described in (–31). NLPR1 has evolved to cleave itself, constitutively, at a specific C terminus-proximal site, indicated by a black arrowhead. The resulting two fragments of NLPR1 remain noncovalently associated in a functionally inactive state. This is denoted by a vertical cylinder between the ZU5 and UPA domains. One of bacterial pathogens that can be detected by NLRP1 is Bacillus anthracis, the cause of anthrax. B. anthracis secretes the “Lethal Factor” (LF), a protease that can enter mammalian cells. Once inside the cell’s cytosol, LF cleaves (a red arrowhead in the diagram) the previously self-cleaved NLPR1. The LF-mediated cleavage of NLPR1 yields the Nt-Leu residue (Fig. 2A). The Arg/N-degron pathway recognizes Nt-Leu, processively destroys this subunit of self-cleaved NLPR1 but spares the associated small subunit UPA-CARD (a former Ct-domain of NLPR1, before its self-cleavage). The released small subunit assembles into an inflammasome complex, whose functions include the activation of caspase-1 (–31).

Fig. 4.

Summary of known functions of the Arg/N-degron pathway. See the main text for references and details.

Fig. 5.

Steric shielding of an Nt-acetylated Nt-residue (Ac/N-degron) of a subunit in a protein complex. Shown here is a part of the crystal structure of a complex between the HCN1 and CUT9 subunits of the fission yeast Schizosaccharomyces pombe APC/C Ub ligase (59). In this structure, the Nt-acetylated Nt-AcMet residue of HCN1 is buried in a chamber formed by the CUT9 subunit. The Nt-region of HCN1 is shown in blue as a stick model. CUT9 is depicted as a cut-out surface representation, to show the chamber’s interior (59). In 2013, Shemorry and Hwang showed that HCN1, when expressed in S. cerevisiae in the absence of CUT9, is a short-lived substrate of the Ac/N-degron pathway (55). In contrast, HCN1 is long-lived upon its coexpression with CUT9 (55).

Fig. 6.

Supramolecular “Chelator–GID” structure of the S. cerevisiae GID ubiquitin ligase. As shown by Schulman, Sherpa, Chrustowicz, and colleagues, this form of GID is particularly efficacious vis-à-vis specific GID substrates such as the Nt-Pro-bearing FBP1 gluconeogenic enzyme, a homotetramer (15, 74). In the enzyme–substrate complex, two GID4 (Pro/N-recognin) subunits of Chelator–GID interact with two (out of four) Nt-Pro residues (denoted as “P”) of the FBP1 homotetramer. A stronger interaction, owing to a chelating effect, increases the efficacy of FBP1 polyubiquitylation by Chelator–GID (15, 74).