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The N-end rule pathway: emerging functions and molecular principles of substrate recognition

Key Points

  • The N-end rule defines the destabilizing activity of a given amino-terminal residue and its post-translational modification. Recognition components (N-recognins) of the N-end rule pathway recognize destabilizing N-terminal amino acids as an essential element of specific N-terminal degrons (N-degrons).

  • A functional N-degron is typically composed of a destabilizing N-terminal residue, an internal Lys residue (the site of polyubiquitylation) and an unstructured N-terminal extension. N-degrons can be generated through endoproteolytic cleavage of polypeptides, which exposes embedded N-degrons at the N termini of C-terminal fragments, or through the post-translational modification of pro-N-degrons, including their deamidation, oxidation, arginylation and acetylation.

  • In mammals, N-recognins characterized by the UBR box bind N-degrons and subsequently induce ubiquitylation and proteasomal degradation, whereas N-recognins in bacteria mediate proteolysis without ubiquitin-like molecules.

  • UBR1 (ubiquitin ligase N-recognin 1)-type N-recognins recognize type 1 and type 2 N-degrons through two distinct sites, the UBR box and the N-domain. The UBR box is a zinc-finger domain that binds a positively charged type 1 residue through a negatively charged, shallow groove. The N-domain appears to have evolutionarily originated from the bacterial N-recognin ClpS which binds a bulky hydrophobic type 2 residue through a deep hydrophobic pocket, within which the N-terminal side chain of the substrate is completely buried.

  • In Saccharomyces cerevisiae, Ubr1 of the N-end rule pathway and ubiquitin-fusion degradation 4 (Ufd4) of the UFD pathway form a complex and synergistically mediate ubiquitylation for both pathways. In complex with Ubr1, Ufd4 functions as an E4-like processivity-enhancing cofactor for Lys48-linked ubiquitylation by Ubr1 after Ubr1 recognizes a substrate.

  • In S. cerevisiae, an acetylated N-terminal residue, including the retained initiator Met, can act as an N-degron, thereby functioning as an alternative signal to initiate the N-end rule pathway. The Doa10 E3 ligase, in concert with the ubiquitin carrier 6 (Ubc6) or Ubc7 E2 enzymes, functions as a new type of N-recognin that mediates the polyubiquitylation of acetylated N-degrons.

Abstract

The N-end rule defines the protein-destabilizing activity of a given amino-terminal residue and its post-translational modification. Since its discovery 25 years ago, the pathway involved in the N-end rule has been thought to target only a limited set of specific substrates of the ubiquitin–proteasome system. Recent studies have provided insights into the components, substrates, functions and structural basis of substrate recognition. The N-end rule pathway is now emerging as a major cellular proteolytic system, in which the majority of proteins are born with or acquire specific N-terminal degradation determinants through protein-specific or global post-translational modifications.

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Figure 1: The hierarchical structures of the N-end rule pathway.
Figure 2: Cooperative targeting by the N-end rule pathway and the UFD pathway.
Figure 3: Structures of N-recognins.
Figure 4: Post-translational modifications that generate N-degrons.
Figure 5: A broad range of substrates and functions.

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Acknowledgements

We are grateful to T. Tasaki for his help throughout the preparation of the manuscript, D. H. Han for editorial assistance, K. Gehring and H. K. Song for critical reading the manuscript, and K. A. Kim for discussions about the N-end rule pathway in the endoplasmic reticulum. Work in the authors' laboratories was supported by grants from the US National Institutes of Health (HL083365 to Y.T.K.), the World Class University (R31-2008-000-10103-0 to Y.T.K.) and the World Class Institute (WCI 2009-002 to B.Y.K.) through the National Research Foundation funded by the Ministry of Education, Science and Technology, Republic of Korea.

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Glossary

E3 ligases

Proteins that recognize specific substrates and accelerate the transfer of ubiquitin from E2 conjugating enzymes to these substrates.

Ubiquitin

A protein with a size of 76 amino acids that exists in all eukaryotic cells and can be conjugated to Lys residues of substrates to provide a secondary degradation signal for the proteasome.

Tertiary destabilizing residues

Amino-terminal degradation determinants that can be recognized and bound by N-recognin through two distinct modifications, such as deamidation and arginylation.

Secondary destabilizing residues

Amino-terminal degradation determinants that can be recognized and bound by N-recognin through a single modification, such as arginylation.

Primary destabilizing residue

The amino-terminal residue of an N-end rule substrate, which acts as the N-terminal degradation determinant without further modification.

N-domain

The second substrate-recognition domain conserved in UBR1-type N-recognins, which binds to type 2 degrons and shares a similarity in the secondary structure with the ClpS N-recognin of bacteria.

Pro-N-degrons

Precursors of amino-terminal degrons (N-degrons), the modification or N-terminal exposure of which can create N-degrons through endoproteolytic cleavage.

Met aminopeptidase

(MetAP). Catalyses the removal of the amino-terminal Met residue when the second residue has a small-sized side chain (for example, Gly, Pro, Ala, Ser, Thr or Cys), and thus exposes the residues at position two at the N terminus.

E4 enzyme

A protein that promotes the processivity of polyubiquitylation by E3 without affecting substrate specificity.

Tetrahedral coordination

Fourfold bonding of a central ion through non-covalent interactions with residues in its proximity, such as a zinc ion interacting with two Cys and two His residues to form four non-covalent contacts.

Salt bridge

A combination of hydrogen bonding and electrostatic interactions in which both donor and acceptor atoms are fully charged for a hydrogen bond.

van der Waals packing

Tight packing of residues owing to weak attractive forces arising from the fluctuations in electron distribution around the nuclei of less-electronegative atoms.

Rotamer

A single side-chain conformation, which represents one dihedral-angle of all possible rotational isomers.

Steric clash

Occurs when two atoms are placed closer than the sum of their atomic radii, thus preventing them from occupying the same volume.

Effector caspases

Activated caspases that are produced from inactive pro-caspases through cleavage by initiator caspases and which subsequently cleave protein substrates to induce the apoptotic process.

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Sriram, S., Kim, B. & Kwon, Y. The N-end rule pathway: emerging functions and molecular principles of substrate recognition. Nat Rev Mol Cell Biol 12, 735–747 (2011). https://doi.org/10.1038/nrm3217

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