Abstract
The ubiquitin-proteasome system is the canonical pathway for protein degradation in eukaryotic cells. 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 intrinsic stabilities. Further, there are multiple means by which substrates are targeted to the proteasome, and these differences could also affect the proteasome's ability to unfold and degrade substrates. Herein we investigate how the fate of GFP variants of differing intrinsic 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, whereas 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. Difficult-to-unfold substrates are released and re-engaged multiple times, with removal of the degradation initiation region providing an alternative clipping pathway that precludes unfolding and degradation; the UBL degron favors degradation of even difficult-to-unfold substrates, whereas the Ub4 degron favors clipping. Finally, we show that the ubiquitin receptor Rpn13 is primarily responsible for the enhanced ability of the proteasome to degrade stable UBL-tagged substrates. Our results indicate that the choice of targeting method and reporter protein are critical to the design of protein degradation experiments.
Highlights
The ubiquitin–proteasome system (UPS) is responsible for the bulk of protein degradation in eukaryotic cells
The Matouschek laboratory used a circular permutant of GFP to show that Rpn10 and to a lesser extent Rpn13 were the only receptors used by Ub4, whereas Rpn1 and Rpn13 were the receptors of choice for the UBL domain [18]
By using substrates containing GFP variants of different stabilities with WT and receptor-mutant proteasomes and using gel-based assays that can detect partial degradation, we show here that UBL-targeted substrates are degraded more efficiently than Ub4-targeted substrates and that this difference seems to be correlated with reliance on the Rpn13 receptor
Summary
We used two previously established targeting systems, the linear Ub4 modification, with four nonhydrolyzable ubiquitins connected by short linkers, and the ubiquitin-like (UBL) domain from yeast Rad, both attached to the N terminus. In both cases, addition of an unstructured initiation region. By using substrates containing GFP variants of different stabilities with WT and receptor-mutant proteasomes and using gel-based assays that can detect partial degradation, we show here that UBL-targeted substrates are degraded more efficiently than Ub4-targeted substrates and that this difference seems to be correlated with reliance on the Rpn receptor
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