Mode of Protein Targeting to the Proteasome Determines Substrate Fate

Abstract

The Ubiquitin Proteasome System (UPS) is the canonical pathway for protein degradation in eukaryotic cells. Proteins targeted for degradation are first tagged with a polyubiquitin chain through a series of enzyme‐catalyzed reactions before degradation. After polyubiquitination, the 26S proteasome hydrolyzes ATP to processively unfold substrates before they are degraded in the 20S core particle. Not all substrates are degraded well by the proteasome, and many factors underlying the proteasome’s unfolding ability remain unknown. We set out to investigate the role of targeting by comparing a linear Ub4 proteasome‐binding tag with the UBL domain from yeast Rad23, which is also commonly used in degradation experiments. We found that the UBL domain allows for degradation of stable sGFP‐containing substrates, while the Ub4 tag does not. Replacing sGFP with a less stable variant allows degradation in either case. Our results suggest that different proteasome‐binding tags, which are presumably recognized by different ubiquitin receptors, can lead to different proteasome unfolding abilities. Support or Funding Information CEB is a Beckman Scholar. This material is based upon work supported by the National Science Foundation under Grant No. 1515229 to DAK.