Mode of targeting to the proteasome determines GFP fate [Enzymology]

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 ...

Source: Journal of Biological Chemistry - November 20, 2020 Category: Chemistry Authors: Christopher Eric Braganca, Daniel Adam Kraut Tags: Protein Synthesis and Degradation Source Type: research