Harnessing the ubiquitin code to respond to environmental cues

Abstract

Ubiquitination is an essential post-translational signal that allows cells to adapt and respond to environmental stimuli. Substrate modifications range from a single ubiquitin molecule to complex polyubiquitin chains, where diverse chain topologies constitute a code that is utilized to modify the functions of proteins in numerous cellular signalling pathways. Diverse ubiquitin chain topologies are generated by linking the C-terminus of ubiquitin to one of seven lysine residues or the N-terminal methionine 1 residue of the preceding ubiquitin. Cooperative action between a large array of E2 conjugating and E3 ligase enzymes supports the formation of not only homotypic ubiquitin chains but also heterotypic mixed or branched chains. This complex array of chain topologies is recognized by proteins containing linkage-specific ubiquitin-binding domains and regulates numerous cellular pathways. Although many functions of the ubiquitin code in plants remain unknown, recent work suggests that specific chain topologies are associated with particular molecular processes. Deciphering the ubiquitin code and how plants utilize it to cope with the changing environment is essential to understand the regulatory mechanisms that underpin myriad stress responses and establishment of environmental tolerance.

Keywords: E2 enzyme; E3 ligase; chain topology; plant stress responses; proteasome; ubiquitin.

Figure 1. The mechanistic basis of linkage-specific ubiquitin chain formation by E2 and E3 enzymes

Polyubiquitination of substrates involves multiple steps and can involve multiple E2 enzymes that mono- or polyubiquitinate substrates. (A) Some E2–E3 combinations can only monoubiquitinate the substrate (S). (B) Linkage-specific ubiquitin chains are generated by several specialized mechanisms. After monoubiquitination of the substrate by Ube2C, the E2 enzyme Ube2S is recruited and associates with both a donor and acceptor ubiquitin. Whereas the donor ubiquitin is bound via both a thioester and non-covalent interactions, the acceptor ubiquitin is bound near its K11 residue through electrostatic interactions. Ube2S then facilitates a reaction in which K11 from the acceptor ubiquitin attacks the thioester bond between Ube2S and the donor ubiquitin. This results in formation of K11-linked di-ubiquitin that is then utilized to elongate ubiquitin chains of the substrate. (C) By contrast, Ube2N utilizes the tightly bound E2-like subunit Ube2V1 (a pseudo E2 that lacks the conserved cysteine residue critical for the catalytic activity of E2 enzymes) to favourably position the K63 side chain of the incoming ubiquitin, whereas Ube2K interacts with a tyrosine residue near K48 of the acceptor ubiquitin to promote K48-linked chain formation (D).

Figure 2. Sequential addition versus en bloc ubiquitination by HECT-type E3 ligases

The E2 binds to the HECT-E3 domain and transfers ubiquitin to the active site cysteine via a thioester bond from which it is transferred to the substrate protein. HECT domains use different mechanisms of chain elongation either by transferring individual ubiquitin molecules to a growing substrate-linked chain (A) or by pre-assembling chains on its active site cysteine before transferring the whole chain to a substrate (B).

Figure 3. The ubiquitin code is recognized by proteins containing single or multiple ubiquitin-binding domains (UBDs)

UBDs are structurally and functionally diverse, reflecting the diversity of the ubiquitin code. Based on their structures, UBDs differ in the cooperative binding of ubiquitin. For example, a combination of two UBDs can be used to specifically recognize K63- (A) or K48-linked (B) chains, while a single zinc finger-containing UBDs recognizes M1-linked di-ubiquitin (C).