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.
Takeuchi
Junko
J
Department of Microbiology and Immunology, University of California, San Francisco, CA 94143, USA.
Chen
Hui
H
Coffino
Philip
P
D000818:Animals; D001667:Binding, Competitive; D002352:Carrier Proteins / Q000737:chemistry*; D004305:Dose-Response Relationship, Drug; D049452:Green Fluorescent Proteins / Q000737:chemistry; D009955:Ornithine Decarboxylase / Q000737:chemistry*; D010455:Peptides / Q000737:chemistry*; D010957:Plasmids / Q000378:metabolism; D046988:Proteasome Endopeptidase Complex / Q000737:chemistry*; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry*; D025801:Ubiquitin / Q000737:chemistry
D002352:Carrier Proteins; D010455:Peptides; C122019:RPN10 protein, S cerevisiae; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; D025801:Ubiquitin; D049452:Green Fluorescent Proteins; D046988:Proteasome Endopeptidase Complex; C061553:ATP dependent 26S protease; D009955:Ornithine Decarboxylase