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 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, 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. 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 while 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.

Linkage reprogramming by tailor-made E3s reveals polyubiquitin chain requirements in DNA-damage bypass

“…A contribution to proteasomal degradation has also been reported for K63-linked chains ( Ohtake et al., 2018 ; Saeki et al., 2009 ). Linear, i.e., methionine (M)1-linked chains are assembled by a single E3, linear ubiquitin chain assembly complex (LUBAC), and they are best known for their cooperation with K63 chains in the inflammatory response ( Rieser et al., 2013 ; Rittinger and Ikeda, 2017 ; Tokunaga et al., 2009 ), but they have also been used to target proteins for proteasomal degradation ( Bragança and Kraut, 2020 ).…”

The 26S Proteasome Switches between ATP-Dependent and -Independent Mechanisms in Response to Substrate Ubiquitination

“…ATPγS causes the proteasome to stall in s3-like substrate processing states [ 21 ], preventing switching into the s1 substrate-accepting state and thus halting proteasomal engagement and degradation. For some substrates, stalling allows an ATP-independent clipping mechanism to predominate, and the ATPγS-stalled proteasome can also degrade some intrinsically disordered proteins [ 15 , 22 ]. ATPγS considerably slowed degradation of the ubiquitinated-Rpn4-containing substrate and reduced the unfolding ability from 2.1 ± 0.3 to 0.6 ± 0.2 (64% ± 6% of engagements stalled and led to fragment formation) in the presence of NADPH.…”

“…Rpn4- and yODC-containing substrates were overexpressed, purified (including removal of the His-SUMO tag), labeled with sulfo-cyanine5 maleimide (Lumiprobe, Hunt Valley, MD, USA), and repurified by gel filtration as described previously [ 14 ]. UBL-sGFP-102-His 6 was purified as described previously [ 15 ]. Sequences and molecular weights are given in Supplemental Table S1 .…”

“…Reactions were analyzed via SDS-PAGE and fluorescence detection using a Typhoon FLA 9500 (Cytiva, Marlborough, MA, USA). For reconstitution assays, 19S and 20S proteasomes, purified as described previously, were reconstituted in a 3:1 ratio (75 nM:25 nM) for 5 min at 30 °C before beginning the assay [ 15 ]. Samples from reconstitution assays were also removed to ice at the indicated times, run on a 3.5% native gel, and visualized using Suc-LLVY-AMC in the presence of 0.02% SDS [ 17 ].…”

Ellagic Acid Triggers the Necrosis of Differentiated Human Enterocytes Exposed to 3-Nitro-Tyrosine: An MS-Based Proteomic Study

“…Finally, the process of protein repair was also affected by the exposure to 3-NT+EA. Proteins related to the proteasome complex involved in the proteolytic degradation of most intracellular proteins [ 60 ] were found in higher abundance in the presence of 3-NT+EA-treated cells. Other proteins involved in post-replication repair and DNA damage checkpoint activation, such as RPA3 [ 61 ], were also increased in quantity as compared to 3-NT-treated cells.…”