Recognition and processing of ubiquitin-protein conjugates by the proteasome

Abstract

The proteasome is an intricate molecular machine, which serves to degrade proteins following their conjugation to ubiquitin. Substrates dock onto the proteasome at its 19-subunit regulatory particle via a diverse set of ubiquitin receptors and are then translocated into an internal chamber within the 28-subunit proteolytic core particle (CP), where they are hydrolyzed. Substrate is threaded into the CP through a narrow gated channel, and thus translocation requires unfolding of the substrate. Six distinct ATPases in the regulatory particle appear to form a ring complex and to drive unfolding as well as translocation. ATP-dependent, degradation-coupled deubiquitination of the substrate is required both for efficient substrate degradation and for preventing the degradation of the ubiquitin tag. However, the proteasome also contains deubiquitinating enzymes (DUBs) that can remove ubiquitin before substrate degradation initiates, thus allowing some substrates to dissociate from the proteasome and escape degradation. Here we examine the key elements of this molecular machine and how they cooperate in the processing of proteolytic substrates.

Figure 1

The ubiquitin-proteasome pathway for protein breakdown. Ubiquitin in green, substrate in yellow. A ubiquitin chain, synthesized via a cascade of E1, E2, and E3 enzymes, is thought to be the predominant signal for substrate recognition by the proteasome. For some substrates, however, modification by a ubiquitin chain is not obligatory (see text for details).

Figure 2

Structure of the proteasome holoenzyme. The lid is highlighted in green, the base in purple, and the CP in orange. The position of the joint between the lid and base is only an estimate. The image was generated by averaging of electron micrographs of negatively stained X. leavis proteasomes. Modified from ref. , with permission.

Figure 3

Gallery of proteasome core particle images. Upper left, Surface representation of the CP, showing its organization into 4 heptameric rings of subunits. The CP is shown along its 2-fold symmetry axis. Each subunit and its symmetry mate were painted in the same color except for b1 and b1′. Upper right, free, closed-channel state of the CP showing sequestration of the proteolytic sites (painted in red) and the closed channel. A medial section is shown, with the slice surface in green. A bracket indicates the approximate position of the channel entry port as seen in the open state. Adapted from ref. with permission. (C) α ring of the free CP as seen from the side where RP and PA26 bind, with α pockets of the CP in blue. Channel is closed. (D) α ring with bound PA26 C-termini (yellow) and the channel in the open state. Structural coordinates taken from 1RYP (closed) and 1FNT (open), from refs. and .

Figure 4

Ubiquitin receptors of S. cerevisiae and humans. Authentic proteasome subunits are classified as intrinsic receptors, whereas proteins reversibly associating with the proteasome are termed shuttling receptors. The form of each receptor protein seen in humans is depicted, except for Ddi1, whose human homolog as no UBA domain. Schematics for canonical members of each class are shown, and, apart from ubiquitin, all proteins and domains are drawn to scale. UIM, UBA, STI1, VWA and PB1 are SMART domains (http://smart.embl-heidelberg.de/). RVP is a Pfam domain, and is encompassed by the slightly larger aspartyl protease Pfam domain. In all cases but two, UBL corresponds to the SMART UBQ domain. UBL domains indicated for scDdi1 and hNub1 are not recognized by SMART, but a requirement for the UBL in proteasome binding has been demonstrated (123, 131, 158). The ZnF domain refers to two different types of zinc finger domains, and specifically ZnF(AN1) in AIRAPL and ZnF(ZZ) in p62. The PRU domain is from refs. and . Aliases and accession numbers are given in a Supplementary Table.

Figure 5

Gallery of ubiquitin-ubiquitin receptor complexes mediating substrate recognition by the proteasome. (A) Rpn10 (PDB code 1WR1), (B) Rpn13 (PDB codes 2Z59 and 2R2Y), (C) Dsk2 (PDB code 1YX5). Figure adapted from ref. with permission.