Mode of targeting to the proteasome determines GFP fate

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. 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. The ubiquitin–proteasome system (UPS) is responsible for the bulk of protein degradation in eukaryotic cells. Proteins to be degraded are typically polyubiquitinated on one or more lysines by the action of E1, E2, and E3 enzymes. The polyubiquitin chain is recognized by one of three ubiquitin receptors on the 19S subunit of the proteasome, Rpn1, Rpn10, or Rpn13, or by ubiquitin shuttle proteins that bind to both polyubiquitin and one of the ubiquitin receptors. The substrate is then engaged by the ATP-dependent Rpt motor proteins at an unstructured initiation region and translocated into the 20S core particle's central degradation chamber. The polyubiquitin chain passes by the deubiquitinase Rpn11 during translocation, which removes the chain, allowing ubiquitin to be recycled for additional rounds of protein targeting. Although recent advances have led to the understanding of many of the details of substrate recognition and unfolding, it remains unclear why three ubiquitin receptors (and multiple adaptors) are necessary or what controls whether a protein targeted to the proteasome by ubiquitination is successfully unfolded or escapes degradation. 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However, there are to using GFP as a model GFP an stable of a from the by ATP-dependent to a stable and the is T.A. R.T. unfolding of a protein by the PubMed Scopus Google Scholar). as an of degradation have been both in cells and in vitro K.E. ubiquitination controls the unfolding ability of the 2016; PubMed Scopus Google these are frequently most at the other circular of GFP that unfold in a have been to be degraded by the ATP-dependent without T.A. R.T. unfolding of a protein by the PubMed Scopus Google Scholar). The proteasome been to be even more P. S. S. C. Matouschek A. ATP-dependent in their ability to unfold 2009; PubMed Scopus Google Scholar) and of even difficult-to-unfold substrates GFP without A. Anderson R.T. Smith D.M. The proteasomal a to unfold PubMed Scopus Google Scholar). However, in some GFP unfolding and degradation in cells the in to the proteasome's activity R.M. Li protein is a in ubiquitin–proteasome 2001; PubMed Scopus Google Scholar, A. Dantuma N.P. P. A activity for the in proteasomal 2009; PubMed Scopus Google Scholar, C. A. The protein substrates for degradation by the 2019; PubMed Scopus Google Scholar). a understanding of GFP unfolding by the proteasome will the of a of experiments. We that substrate ubiquitination the proteasome's unfolding ability and that proteasomal ubiquitin receptors with Rpn13 the K.E. ubiquitination controls the unfolding ability of the 2016; PubMed Scopus Google Scholar, J.D. A. J. receptors are for of the proteasome's unfolding 2019; 9 PubMed Scopus Google Scholar). Herein we to determine whether the degradation of GFP is by the mode of targeting to the proteasome, which ubiquitin receptors are We used two targeting the linear Ub4 with by and the domain from yeast Rad23, both to the both of an unstructured initiation region at the of a protein been to to degradation by the proteasome T. S. S. Matouschek A. the of the proteasome PubMed Scopus Google Scholar). been to be the receptor for S. G. N. de S. J. receptor for substrate and by the 2016; PubMed Scopus Google Scholar), and Rpn13 are to be the receptors for substrates The recognition of proteins by the Sci. 2016; PubMed Scopus Google Scholar). the Matouschek used a circular of GFP to show that and to a Rpn13 the receptors used by whereas and Rpn13 the receptors of choice for the UBL domain K. C. T. S. Matouschek A. The proteasome 19S and its ubiquitin receptors a recognition for Commun. 2020; PubMed Scopus Google Scholar). However, a substrate it unclear to what the proteasome's unfolding ability is by the targeting mode or receptor using substrates GFP variants of different stabilities with and and using that we show that substrates are degraded more substrates and that to be with on the Rpn13 We the ability of the proteasome to degrade a substrate of GFP with a linear Ub4 on the and a unstructured of a The substrate with yeast proteasome, and the on an and and using the of GFP is unfolded or it degradation of as determined by quantifying the However, there of the and as been with other substrates K.E. ubiquitination controls the unfolding ability of the 2016; PubMed Scopus Google Scholar, T.A. R.T. unfolding of a protein by the PubMed Scopus Google Scholar). could the unstructured is to unfolding or the proteasome it is to unfold and the initiation degradation of some proteasomal substrates K. C. T. S. Matouschek A. The proteasome 19S and its ubiquitin receptors a recognition for Commun. 2020; PubMed Scopus Google Scholar, T. S. S. Matouschek A. the of the proteasome PubMed Scopus Google Scholar), we the with an the and of and clipping the of degradation and the other of with a circular of GFP which the is the of the led to degradation of the substrate with clipping with the that the proteasome to unfold the stable the that the linear Ub4 is of targeting proteins to the proteasome, the ability to successfully unfold and degrade the substrates on their stabilities. we that the substrate bind to the proteasome, it to the degradation of a substrate clipping of the substrate and degradation of the substrate both by proteasome inhibitors A and However, the of polyubiquitin degradation of clipping of that clipping does on ubiquitin and We that clipping of could be by either the 20S or proteasome, both 20S core or proteasome by the of 19S to 20S core the substrate the substrate by 20S proteasome degraded 19S with the stable enhanced GFP led to of clipping and degradation and the linear Ub4 proteins to the proteasome, their unfolding and degradation on their stabilities. substrates are stable to be unfolded they are in a and the unstructured initiation and we the Ub4 targeting on with the UBL domain from yeast The proteasome to degrade substrate been H. G. Matouschek A. to the proteasome proteolysis a J. 2016; PubMed Scopus Google with clipping A and a that the of of protein the of of A also that an unstructured to as an initiation from the to clipping of the substrates a pathway with the substrates, with the stable the and degradation and the of clipping that the substrate degraded without the of or of that substrate also as a on a of differences and the of degradation of a GFP protein with a UBL then with a Ub4 indicating that the UBL domain is to the unfolding and degradation of the substrate by the degraded protein be and model for degradation of The substrate be a in a or be the UBL and and then the is degraded to the which either be unfolded and degraded or be released and it is from the model to the from and For and the of for H. G. Matouschek A. to the proteasome proteolysis a J. 2016; PubMed Scopus Google Scholar) used as the and to as determined for Ub4 from degradation by the a PubMed Scopus Google Scholar) differences are to to for a of substrate that remains the and to the as as the that for degradation with proteasome and in which is 9 or at the of the and of from as in show and are with of show of been that for the proteasomal with led to a in GFP degradation A. Anderson R.T. Smith D.M. The proteasomal a to unfold PubMed Scopus Google Scholar). 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J. P. of proteasome on substrate PubMed Scopus Google Scholar). The ability to and the substrate degradation it to degrade substrates the is to the initiation region to many proteins ubiquitination occurs on the initiation that ubiquitin on the substrate be to and the ability of the proteasome to unstructured initiation as a the substrate from Ub4 more to the proteasome UBL H. G. Matouschek A. to the proteasome proteolysis a J. 2016; PubMed Scopus Google Scholar, K. Matouschek A. in vitro proteasome activity 2018; PubMed Scopus Google Scholar). However, the in model a on that the proteasome degrade the substrate with clipping. that a substrate is degraded by either proteasome, there are also differences in the and unfolding Ub4 and UBL substrates be at the proteasome, and even unfolding We to whether one or more of the intrinsic proteasomal ubiquitin receptors responsible for the proteasome's ability to degrade substrates with UBL Ub4 targeting. The Matouschek that the linear Ub4 degron more the is recognized and degraded by both and Rpn13 whereas the UBL substrate is degraded even three receptors are or Rpn13 for degradation K. C. T. S. Matouschek A. The proteasome 19S and its ubiquitin receptors a recognition for Commun. 2020; PubMed Scopus Google Scholar). determine the for a we used or which is degraded with the UBL and with with the Ub4 and targeted the Ub4 signal, proteasome receptor and of which to ubiquitin J.D. A. J. receptors are for of the proteasome's unfolding 2019; 9 PubMed Scopus Google the of clipping in the of the of degradation of A and and one receptor and clipping effects on degradation that of the receptors are of at some degradation of of the receptors are to targeting the UBL domain more either or Rpn13 clipping in the of degradation and Surprisingly, degradation that of the UBL domain to be or The effects of even more proteasome whereas and proteasome of unfolding and with degradation and clipping. these the UBL substrate into the degraded Ub4 indicating that Rpn13 is primarily responsible for the enhanced unfolding and degradation of substrates, the of and is to for the of ability to bind the UBL We that the to degrade to a of substrate the substrate degraded by the proteasome with an from without of GFP is commonly used as a model substrate to study protein degradation. we show that both the ubiquitin and the GFP stability are for whether GFP is successfully unfolded and degraded or whether degradation is substrates with the stability are degraded more substrates. unfolding ability is at in by the Rpn13 ubiquitin proteins have been to the proteasome multiple by the to the 20S core the activity of the proteasome, and the proteasome's ability to unfold substrates K.E. ubiquitination controls the unfolding ability of the 2016; PubMed Scopus Google Scholar, J.D. A. J. receptors are for of the proteasome's unfolding 2019; 9 PubMed Scopus Google Scholar, A. N. M. proteins the proteasomal by to or PubMed Scopus Google Scholar, A. proteins the proteasome by to which 20S 2009; PubMed Scopus Google Scholar). effects have been for substrates UBL their ability to bind to many of the receptors that bind UBL are to proteasomal degradation of at sGFP-containing substrates is by unfolding, is to be responsible for the of UBL targeting. We proposed that of proteins to Rpn13 the proteasome's unfolding ability by the from the to in the model of to the unfolding K.E. ubiquitination controls the unfolding ability of the 2016; PubMed Scopus Google Scholar, J.D. A. J. receptors are for of the proteasome's unfolding 2019; 9 PubMed Scopus Google Scholar). The could the UBL Ub4 results we that Ub4 substrates primarily on Rpn10, whereas UBL substrates primarily on Rpn13 K. C. T. S. Matouschek A. The proteasome 19S and its ubiquitin receptors a recognition for Commun. 2020; PubMed Scopus Google Scholar). with of polyubiquitin from Rpn13 been to multiple turnover degradation by the proteasome, that of Rpn13 the proteasome from to the for additional rounds of substrate K. C. T. S. Matouschek A. The proteasome 19S and its ubiquitin receptors a recognition for Commun. 2020; PubMed Scopus Google Scholar). alternative is that differences Ub4 and UBL from the to the from its receptor during a UBL domain be which more during the However, the UBL domain more to the proteasome Ub4 H. G. Matouschek A. to the proteasome proteolysis a J. 2016; PubMed Scopus Google Scholar, K. Matouschek A. in vitro proteasome activity 2018; PubMed Scopus Google Scholar), been why many ubiquitin receptors and shuttle proteins for proteasomal Our results that shuttle proteins be used to proteins to the proteasome by to Rpn13 and an enhanced unfolding whereas proteins are to be degraded without by to it been that there is at one additional ubiquitin or UBL receptor on the proteasome, even a degrade a substrate K. C. T. S. Matouschek A. The proteasome 19S and its ubiquitin receptors a recognition for Commun. 2020; PubMed Scopus Google Scholar). Our results indicate that an additional ubiquitin or UBL receptor it does unfolding of difficult-to-unfold substrates as Our results also indicate that UPS substrates the degron and unstructured initiation is different of the protein be degraded more the initiation region and are one in the allows degraded protein to be to the proteasome, that a is to degradation of the However, is by the ability of the proteasome to unstructured initiation in a which of degradation. initiation which are degradation is T. J.M. Matouschek A. A initiation region in protein its proteasomal Commun. 2020; PubMed Scopus Google Scholar), be both for degradation and for of the initiation We have ATP-dependent from studies of as T.A. R.T. into and 2016; PubMed Scopus (148) Google Scholar, T.A. R.T. protein unfolding and 2018; PubMed Scopus Google Scholar), and of what we have to the proteasome as there are also to degrade GFP in GFP and then the Our results with results for the proteasome A. Anderson R.T. Smith D.M. The proteasomal a to unfold PubMed Scopus Google indicate that the proteasome does have and there is of a GFP that is that the proteasome the unfolded substrate more the that be for to with we have that the proteasome a unfolding ability unfolding and ATP-dependent P. S. S. C. Matouschek A. ATP-dependent in their ability to unfold 2009; PubMed Scopus Google Scholar) and that proteasome an even unfolding ability yeast proteasome of a a and unfolding E. A. K. T. Matouschek A. and of proteasomal 2012; PubMed Scopus Google Scholar). that the to the proteasome an that of the it most that the unfolding ability of the proteasome, and its to on is to a of degraded substrates. Finally, these results have for the of GFP as a in and that the global of the UPS or that the fate of it to a circular in one to whether an protein or how the UPS is for degradation of A more stable GFP as could be used to at the ability of the UPS to degrade more substrates, and the of the targeting mechanism a in how of a proteasome is for degradation. in in and in from the Matouschek A by the UBL domain in with a Ub4 domain by A by the from with the from by substrates by with by A by from yeast into are proteasome from or receptor using a of Rpn11 subunit of the 19S as K.E. ubiquitination controls the unfolding ability of the 2016; PubMed Scopus Google Scholar, J.D. A. J. receptors are for of the proteasome's unfolding 2019; 9 PubMed Scopus Google Scholar). 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