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. Here we tested that idea in several ways. Stabilizing or destabilizing a folded domain within substrate proteins changed GAr-mediated intermediate production in the way predicted by the model. A downstream folded domain determined the sites of terminal proteolysis. The spacing between a GAr and a folded domain was critical for intermediate production. Intermediates containing a GAr did not remain associated with proteasomes, excluding models whereby retained GAr-containing proteins halt further processing. The following model is supported: a GAr positioned within the ATPase ring reduces the efficiency of coupling between nucleotide hydrolysis and work performed on the substrate. If this impairment takes place when unfolding must be initiated, insertion pauses and proteolysis is limited to the portion of the substrate that has already entered the catalytic chamber of the proteasome

Proteasome substrate degradation requires association plus extended peptide

To determine the minimum requirements for substrate recognition and processing by proteasomes, the functional elements of a ubiquitin-independent degradation tag were dissected. The 37-residue C-terminus of ornithine decarboxylase (cODC) is a native degron, which also functions when appended to diverse proteins. Mutating the cysteine 441 residue within cODC impaired its proteasome association in the context of ornithine decarboxylase and prevented the turnover of GFP-cODC in yeast cells. Degradation of GFP-cODC with C441 mutations was restored by providing an alternate proteasome association element via fusion to the Rpn10 proteasome subunit. However, Rpn10-GFP was stable, unless extended by cODC or other peptides of similar size. In vitro reconstitution experiments confirmed the requirement for both proteasome tethering and a loosely structured region. Therefore, cODC and degradation tags in general must serve two functions: proteasome association and a site, consisting of an extended peptide region, used for initiating insertion into the protease