Mode of Targeting to the Proteasome Determines GFP Fate

Abstract

The Ubiquitin-proteasome system (UPS) is the canonical pathway for protein degradation in eukaryotic cells. Green fluorescent protein (GFP) is frequently used as a reporter in proteasomal degradation assays. However, there are multiple variants of GFP in use, and these variants have different stabilities. We previously found that the proteasome’s ability to unfold and degrade substrates is enhanced by polyubiquitin chains on the substrate, and that proteasomal ubiquitin receptors mediate this activation. Herein we investigate how the fate of GFP variants of differing stabilities is determined by the mode of targeting to the proteasome. We compared two targeting systems: linear Ub4 degrons and the UBL domain from yeast Rad23, both of which are commonly used in degradation experiments. Surprisingly, the UBL degron allows for degradation of the most stable sGFP-containing substrates, while the Ub4 degron does not. Destabilizing the GFP by circular permutation allows degradation with either targeting signal, indicating that domain stability and mode of targeting combine to determine substrate fate. Finally, we show that the ubiquitin receptor Rpn13 is primarily responsible for the enhanced ability of the proteasome to degrade stable UBL-tagged substrates.

Ubiquitin modulates 26 S proteasome conformational dynamics and promotes substrate degradation

“…observed to facilitate substrate degradation (42,43), yet their effects beyond substrate recruitment, for instance, on degradation initiation, mechanical processing, or the proteasome conformational states are unknown.…”

“…Despite these previous insights, the detailed mechanisms underlying substrate engagement, deubiquitination, mechanical unfolding, and translocation, as well as the role of proteasome conformational transitions for the individual processing steps remain largely elusive. Furthermore, ubiquitin modifications were observed to facilitate substrate degradation ( 42 , 43 ), yet their effects beyond substrate recruitment, for instance, on degradation initiation, mechanical processing, or the proteasome conformational states are unknown.…”