Reconfiguration of the proteasome during chaperone-mediated assembly.

The proteasomal ATPase ring, comprising Rpt1-Rpt6, associates with the heptameric α-ring of the proteasome core particle (CP) in the mature proteasome, with the Rpt carboxy-terminal tails inserting into pockets of the α-ring. Rpt ring assembly is mediated by four chaperones, each binding a distinct Rpt subunit. Here we report that the base subassembly of the Saccharomyces cerevisiae proteasome, which includes the Rpt ring, forms a high-affinity complex with the CP.

Functional asymmetries of proteasome translocase pore.

Degradation by proteasomes involves coupled translocation and unfolding of its protein substrates. Six distinct but paralogous proteasome ATPase proteins, Rpt1 to -6, form a heterohexameric ring that acts on substrates. An axially positioned loop (Ar-Φ loop) moves in concert with ATP hydrolysis, engages substrate, and propels it into a proteolytic chamber.

Dependence of proteasome processing rate on substrate unfolding.

Protein degradation by eukaryotic proteasomes is a multi-step process involving substrate recognition, ATP-dependent unfolding, translocation into the proteolytic core particle, and finally proteolysis. To date, most investigations of proteasome function have focused on the first and the last steps in this process. Here we examine the relationship between the stability of a folded protein domain and its degradation rate.

Ordering an engagement ring.

In this issue of Molecular Cell, Tomko et al. (2010) establish that the six distinct ATPase subunits of the eukaryotic proteasome form a heterohexameric ring and resolve how the subunits are arranged within the ring.

A genetic screen for Saccharomyces cerevisiae mutants affecting proteasome function, using a ubiquitin-independent substrate.

The great majority of proteasome substrates are marked for degradation by the attachment of polyubiquitin chains. Ornithine decarboxylase is degraded by the proteasome in the absence of this modification. We previously showed that this mechanism of degradation was conserved in eukaryotic cells.

Structural elements of the ubiquitin-independent proteasome degron of ornithine decarboxylase.

Mouse ODC (ornithine decarboxylase) is quickly degraded by the 26S proteasome in mammalian and fungal cells. Its degradation is independent of ubiquitin but requires a degradation signal composed of residues 425-461 at the ODC C-terminus, cODC (the last 37 amino acids of the ODC C-terminus). Mutational analysis of cODC revealed the presence of two essential elements in the degradation signal.

Glycine-alanine repeats impair proper substrate unfolding by the proteasome.

Proteasome ATPases unravel folded proteins. Introducing a sequence containing only glycine and alanine residues (GAr) into substrates can impair their digestion. We previously proposed that a GAr interferes with the unfolding capacity of the proteasome, leading to partial degradation of products.

Probing the ubiquitin/proteasome system with ornithine decarboxylase, a ubiquitin-independent substrate.

Ornithine decarboxylase (ODC) is an unusual proteasome substrate-ubiquitin conjugation plays no part in its turnover. It can therefore be used as a probe to distinguish proteasome-mediated actions that do or do not depend on the activity of the ubiquitin system. A 37 residue region of ODC suffices for proteasome interactions, and within this sequence functionally critical residues have been identified.

Proteasomes begin ornithine decarboxylase digestion at the C terminus.

Proteasomes denature folded protein substrates and thread them through a narrow pore that leads to the sequestered sites of proteolysis. Whether a protein substrate initiates insertion from its N or C terminus or in a random orientation has not been determined for any natural substrate. We used the labile enzyme ornithine decarboxylase (ODC), which is recognized by the proteasome via a 37-residue C-terminal tag, to answer this question.

Ubiquitin-independent mechanisms of mouse ornithine decarboxylase degradation are conserved between mammalian and fungal cells.

The polyamine biosynthetic enzyme ornithine decarboxylase (ODC) is degraded by the 26 S proteasome via a ubiquitin-independent pathway in mammalian cells. Its degradation is greatly accelerated by association with the polyamine-induced regulatory protein antizyme 1 (AZ1). Mouse ODC (mODC) that is expressed in the yeast Saccharomyces cerevisiae is also rapidly degraded by the proteasome of that organism.

An easily dissociated 26 S proteasome catalyzes an essential ubiquitin-mediated protein degradation pathway in Trypanosoma brucei.

The 26 S proteasome, a complex between the 20 S proteasome and 19 S regulatory units, catalyzes ATP-dependent degradation of unfolded and ubiquitinated proteins in eukaryotes. We have identified previously 20 S and activated 20 S proteasomes in Trypanosoma brucei, but not 26 S proteasome. However, the presence of 26 S proteasome in T.