Rad23 and Rpn10 Serve as Alternative Ubiquitin Receptors for the Proteasome

Abstract

The selective recognition of ubiquitin conjugates by proteasomes is a key step in protein degradation. The receptors that mediate this step have yet to be clearly defined although specific candidates exist. Here we show that the proteasome directly recognizes ubiquitin chains through a specific subunit, Rpn10, and also recognizes chains indirectly through Rad23, a reversibly bound proteasome cofactor. Both binding events can be observed in purified biochemical systems. A block substitution in the chain-binding ubiquitin interacting motif of RPN10 when combined with a null mutation in RAD23 results in a synthetic defect in protein degradation consistent with the view that the direct and indirect recognition modes function to some extent redundantly in vivo. Rad23 and the deubiquitinating enzyme Ubp6 both bind proteasome subunit Rpn1 through N-terminal ubiquitin-like domains. Surprisingly, Rad23 and Ubp6 do not compete with each other for proteasome binding. Thus, Rpn1 may act as a scaffold to assemble on the proteasome multiple proteins that act to either bind or hydrolyze multiubiquitin chains. The selective recognition of ubiquitin conjugates by proteasomes is a key step in protein degradation. The receptors that mediate this step have yet to be clearly defined although specific candidates exist. Here we show that the proteasome directly recognizes ubiquitin chains through a specific subunit, Rpn10, and also recognizes chains indirectly through Rad23, a reversibly bound proteasome cofactor. Both binding events can be observed in purified biochemical systems. A block substitution in the chain-binding ubiquitin interacting motif of RPN10 when combined with a null mutation in RAD23 results in a synthetic defect in protein degradation consistent with the view that the direct and indirect recognition modes function to some extent redundantly in vivo. Rad23 and the deubiquitinating enzyme Ubp6 both bind proteasome subunit Rpn1 through N-terminal ubiquitin-like domains. Surprisingly, Rad23 and Ubp6 do not compete with each other for proteasome binding. Thus, Rpn1 may act as a scaffold to assemble on the proteasome multiple proteins that act to either bind or hydrolyze multiubiquitin chains. The breakdown of ubiquitin-protein conjugates by the proteasome is a major mechanism for biological regulation in eukaryotes (1Pickart C.M. Cohen R.E. Nat. Rev. Mol. Cell. Biol. 2004; 5: 177-187Crossref PubMed Scopus (611) Google Scholar). The conjugative and degradative machineries are coupled at the ubiquitin chain recognition step. This step, although intensively studied, has remained problematic (1Pickart C.M. Cohen R.E. Nat. Rev. Mol. Cell. Biol. 2004; 5: 177-187Crossref PubMed Scopus (611) Google Scholar, 2Deveraux Q. Ustrell V. Pickart C. Rechsteiner M. J. Biol. 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Among the putative shuttling factors having affinity for both the proteasome and ubiquitin conjugates is a family of proteins exemplified by the Rad23 protein of Saccharomyces cerevisiae (27Hartmann-Petersen R. Seeger M. Gordon C. Trends Biochem. Sci. 2003; 28: 26-31Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar, 28Madura K. Cell Cycle. 2002; 1: 235-244Crossref PubMed Scopus (43) Google Scholar). Proteasome binding by Rad23-like proteins involves their N-terminal ubiquitin-like (UBL) 1The abbreviations used are: UBL, ubiquitin-like; UBA, ubiquitin associated; UIM, ubiquitin interacting motif; GST, glutathione S-transferase; suc, succinyl; AMC, 7-amido-4 methylcoumarin. domains (8Schauber C. Chen L. Tongaonkar P. Vega I. Lambertson D. Potts W. Madura K. Nature. 1998; 391: 715-718Crossref PubMed Scopus (408) Google Scholar, 9Funakoshi M. Sasaki T. Nishimoto T. Kobayashi H. Proc. Natl. Acad. Sci. U. S. 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Cell Biol. 2002; 4: 725-730Crossref PubMed Scopus (383) Google Scholar), and for the Rad23 binding of ubiquitin the the the and domains K.J. Lech P.J. Goh A.M. Wang Q. Howley P.M. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 12694-12699Crossref PubMed Scopus (128) Google Scholar). the the and domains to the binding of the to the proteasome. the of the shuttling A for proteins in degradation is by the that some are in Rad23 and in family M. Sasaki T. Nishimoto T. Kobayashi H. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 745-750Crossref PubMed Scopus (212) Google Scholar, 17Wilkinson C.R. Seeger M. Hartmann-Petersen R. Stone M. Wallace M. Semple C. Gordon C. Nat. Cell Biol. 2001; 3: 939-943Crossref PubMed Scopus (351) Google Scholar, D. Chen L. Madura K. 1999; PubMed Google Scholar). of by has to to the of in S. A. M. C. Mol. Cell. Biol. 2003; PubMed Scopus Google Scholar). with a for Rad23 and in degradation is the that the of Rad23 on the in degradation of and to be S. Pickart C.M. J. 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Full Text PDF PubMed Google Scholar). the proteasome subunit in binding Q. Ustrell V. Pickart C. Rechsteiner M. J. Biol. Chem. 1994; 269: 7059-7061Abstract Full Text PDF PubMed Google Scholar). Rpn10, the of to be that other ubiquitin receptors Nocker S. Sadis S. Rubin D.M. Glickman M. Fu H. Coux O. Wefes I. Finley D. Vierstra R.D. Mol. Cell. Biol. 1996; 16: 6020-6028Crossref PubMed Scopus (357) Google Scholar). are to is in Nocker S. Sadis S. Rubin D.M. Glickman M. Fu H. Coux O. Wefes I. Finley D. Vierstra R.D. Mol. Cell. Biol. 1996; 16: 6020-6028Crossref PubMed Scopus (357) Google Scholar). Surprisingly, the not to the ubiquitin interacting motif to the A H. Lee Y. Glickman M.H. Vierstra R.D. EMBO J. 2001; PubMed Scopus (212) Google Scholar), is for proteasome M.H. Rubin D.M. Coux O. Wefes I. G. W. Finley D. Cell. 1998; Full Text Full Text PDF PubMed Scopus Google This and the that to ubiquitin chains not have to the view that is not a ubiquitin Y.A. Lawson T.G. Velayutham M. 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Cell Biol. 2002; 4: 725-730Crossref PubMed Scopus (383) Google Scholar). the of the in recognition has remained as be that proteasomes when have that chain recognition by other proteasome the chain binding by the in the proteasome in is the of whether has a in protein degradation in vivo. are purified we that proteasomes ubiquitin chains directly the of also indirectly through with in specific the mutation and of either RAD23 or the that both direct and indirect for chain recognition by the proteasome. to and are in I. the of the and and and the J.H. 1999; PubMed Scopus Google the A to the RPN10 the and the of the the with to a at to and a of the A of the RPN10 the of the This with the and of the by and the of the mutation by in a the of and of the with the A. A. R. P. 1994; PubMed Scopus Google Scholar, J.H. 1999; PubMed Scopus Google Scholar). by and of Ubp6 purified as D.S. J. A. B. M. T. H. Finley D. Mol. Cell. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). proteins of Rad23 and Rpn1 in and purified in the of and proteins with as W. F. T. Y. P.J. D.M. Cell. Full Text PDF PubMed Scopus Google Scholar). Rad23 proteins the with subsequently with purified in and and with in The used for the of and of conjugates in the of and and for at and with in the of ubiquitin to in the and purified as S. Gali R.R. Schwickart M. Larsen C.N. Leggett D.S. Muller B. Feng M.T. Tubing F. Dittmar G.A. Finley D. Nat. Cell Biol. 2002; 4: 725-730Crossref PubMed Scopus (383) Google Scholar), and ubiquitin for in a with proteasome and for at of the binding of of and of each Rad23 this the of each protein to the with to the by in a of and and for at and by as S. Gali R.R. Schwickart M. Larsen C.N. Leggett D.S. Muller B. Feng M.T. Tubing F. Dittmar G.A. Finley D. Nat. Cell Biol. 2002; 4: 725-730Crossref PubMed Scopus (383) Google Scholar). Proteasome the and proteasomes purified and as and in the of D.S. J. A. B. M. T. H. Finley D. Mol. Cell. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). the and proteasome Ubp6 purified in the of and D.S. J. A. B. M. T. H. Finley D. Mol. Cell. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). the proteasome purified and in the of and the with proteasome and as and as a in with and the protein and used to the on the to the at of with of Rad23 and as of binding at with of by through and through or at to of as and on and for and at of to the Proteasome by the shuttling is that the proteasome has a intrinsic affinity for conjugates L. Rechsteiner M. Pickart C.M. EMBO J. 2000; PubMed Scopus Google Scholar), and affinity not to be by a direct for ubiquitin binding by the by proteasome S. Gali R.R. Schwickart M. Larsen C.N. Leggett D.S. Muller B. Feng M.T. Tubing F. Dittmar G.A. Finley D. Nat. Cell Biol. 2002; 4: 725-730Crossref PubMed Scopus (383) Google Scholar). have used in conjugates in this to whether Rad23 can the binding to proteasomes of conjugates that not show binding. in of can be when Rad23, the and ubiquitin conjugates are be for the species to that the species is a as of the the of Rad23 as as the the N-terminal of Rad23 and the are as to the the not This that is on the of Rad23 to bind the proteasome and ubiquitin conjugates as conjugates are to proteasomes through a Rad23 the of a the with proteasomes for ubiquitin binding and not both S. cerevisiae and proteasome subunit Rpn1 Rad23 proteasomes through recognition of S. Gali R.R. Schwickart M. Larsen C.N. Leggett D.S. Muller B. Feng M.T. Tubing F. Dittmar G.A. Finley D. Nat. Cell Biol. 2002; 4: 725-730Crossref PubMed Scopus (383) Google Scholar, 11Seeger M. Hartmann-Petersen R. Wilkinson C.R. Wallace M. Samejima I. Taylor M.S. Gordon C. J. Biol. Chem. 2003; 278: 16791-16796Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar, 14Saeki Y. Sone T. Toh-e A. Yokosawa H. Biochem. Biophys. Res. Commun. 2002; 296: 813-819Crossref PubMed Scopus (121) Google Scholar). deubiquitinating enzyme also proteasomes by of and Rpn1 D.S. J. A. B. M. T. H. Finley D. Mol. Cell. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). proteasomes are not with Ubp6 D.S. J. A. B. M. T. H. Finley D. Mol. Cell. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). that Rad23 and Ubp6 may compete with each other for binding to proteasomes and that a of proteasomes may be for this the binding of Rad23 to proteasome in the and of in Ubp6 has no on Rad23 binding. Thus, although Rad23 and Ubp6 both bind proteasomes their do through binding on this we binding as the for Rad23 and Ubp6 bound Rpn1 not binding proteins that are on ubiquitin Rpn1 may a in the proteasome. we the Rad23 binding in Rpn1 a of a S. Gali R.R. Schwickart M. Larsen C.N. Leggett D.S. Muller B. Feng M.T. Tubing F. Dittmar G.A. Finley D. Nat. Cell Biol. 2002; 4: 725-730Crossref PubMed Scopus (383) Google Scholar). with the Rpn1 that specific and affinity for Rad23 S. Gali R.R. Schwickart M. Larsen C.N. Leggett D.S. Muller B. Feng M.T. Tubing F. Dittmar G.A. Finley D. Nat. Cell Biol. 2002; 4: 725-730Crossref PubMed Scopus (383) Google or no specific binding to Ubp6 the that the binding of Ubp6 to Rpn1 is as with Rad23 not are in agreement with the of a binding for has not to the Ubp6 binding on whether the binding for Rad23 and Ubp6 are in of to the Proteasome by in both and Nocker S. Sadis S. Rubin D.M. Glickman M. Fu H. Coux O. Wefes I. Finley D. Vierstra R.D. Mol. Cell. Biol. 1996; 16: 6020-6028Crossref PubMed Scopus (357) Google Scholar, C.R. K. M. Wallace M. W. Gordon C. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar, L. A. J. Biochem. PubMed Scopus Google Scholar). is that can bind ubiquitin chains and that motif is for this H. Sadis S. Rubin D.M. Glickman M. van Nocker S. Finley D. Vierstra R.D. J. Biol. Chem. 1998; 273: 1970-1981Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar, P. Deveraux Q. R.E. Pickart C.M. Rechsteiner M. J. Biol. Chem. 1998; 273: Full Text Full Text PDF PubMed Scopus Google Scholar). the of to bind ubiquitin chains is not to ubiquitin chains Y.A. Lawson T.G. Velayutham M. Zweier J.L. Pickart C.M. Nature. 2002; 416: 763-767Crossref PubMed Scopus (370) Google Scholar). proteasomes purified are in chain binding S. Gali R.R. Schwickart M. Larsen C.N. Leggett D.S. Muller B. Feng M.T. Tubing F. Dittmar G.A. Finley D. Nat. Cell Biol. 2002; 4: 725-730Crossref PubMed Scopus (383) Google Scholar), null have a multiple that do not to the chain-binding motif H. Sadis S. Rubin D.M. Glickman M. van Nocker S. Finley D. Vierstra R.D. J. Biol. 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Commun. 2002; 293: 986-992Crossref PubMed Scopus (114) Google and not the in in proteasomes purified the also for the with ubiquitin of the of not a in the of conjugates This coupled with the of on Rad23 and a in the proteins ubiquitin conjugates the proteasome of the motif and are in is Rad23, and for to has used to for proteasome in the of are to and on the proteasome. have a to the the of the A of is for proteasome H. Lee Y. Glickman M.H. Vierstra R.D. EMBO J. 2001; PubMed Scopus (212) Google Scholar). show a of to H. Sadis S. Rubin D.M. Glickman M. van Nocker S. Finley D. Vierstra R.D. J. Biol. Chem. 1998; 273: 1970-1981Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar). The of Rad23, and the on protein degradation by for synthetic to the mutation and for either RAD23 or are to both the proteins and in protein degradation. to be to at consistent with for proteins in degradation by the The synthetic on and that proteins not in protein degradation. Both are consistent with the in of results that at can be used by proteasomes to ubiquitin chains. and Rad23 have to bind ubiquitin their to chains the proteasome has S. Pickart C.M. J. Biol. Chem. 2003; 278: 8951-8959Abstract Full Text Full Text PDF PubMed Scopus (207) Google and has not in a defined in view is that Rad23 and the proteasome can compete for chain and this view have S. Pickart C.M. J. Biol. Chem. 2003; 278: 8951-8959Abstract Full Text Full Text PDF PubMed Scopus (207) Google Scholar). clearly show that Rad23 can chain binding by purified have not of to binding in to of Rad23 to by are key also that ubiquitin chains with and consistent with the in binding is on Rad23 and results that proteins conjugates the proteasome not in in as The in chain recognition has on the of of chains to Y.A. Lawson T.G. Velayutham M. Zweier J.L. Pickart C.M. Nature. 2002; 416: 763-767Crossref PubMed Scopus (370) Google Scholar). This to the that the of is the proteasome Y.A. Lawson T.G. Velayutham M. Zweier J.L. Pickart C.M. Nature. 2002; 416: 763-767Crossref PubMed Scopus (370) Google Scholar). of this the not to have general on protein in Nocker S. Sadis S. Rubin D.M. Glickman M. Fu H. Coux O. Wefes I. Finley D. Vierstra R.D. Mol. Cell. Biol. 1996; 16: 6020-6028Crossref PubMed Scopus (357) Google Scholar, 5Fu H. Sadis S. Rubin D.M. Glickman M. van Nocker S. Finley D. Vierstra R.D. J. Biol. Chem. 1998; 273: 1970-1981Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar). have the view that the of the may that the ubiquitin function of with that of other factors H. Sadis S. Rubin D.M. Glickman M. van Nocker S. Finley D. Vierstra R.D. J. Biol. Chem. 1998; 273: 1970-1981Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar, 7Finley D. Nat. Cell Biol. 2002; 4: E121-E123Crossref PubMed Scopus (28) Google Scholar). The defect in chain binding by purified proteasomes in of this of the of to to ubiquitin chains is that chains may to a subunit in the that the proteins function as ubiquitin we to the that the of the in are by the of chain-binding factors such as Rad23 and The synthetic the mutation and observed There are of and null Y. Saitoh A. Toh-e A. Yokosawa H. Biochem. Biophys. Res. Commun. 2002; 293: 986-992Crossref PubMed Scopus (114) Google Scholar, 21Chen L. Madura K. Mol. Cell. Biol. 2002; 22: 4902-4913Crossref PubMed Scopus (253) Google Scholar, D. Chen L. Madura K. 1999; PubMed Google Scholar), do not the the major of null the do not to the The the in this although that the are and that the modes are not in This may to that of Chen and Madura L. Madura K. Mol. Cell. Biol. 2002; 22: 4902-4913Crossref PubMed Scopus (253) Google Scholar), observed that of Rad23 to the binding of ubiquitin conjugates to the proteasome in not in L. Madura K. Mol. Cell. 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