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Proteasome activator Blm10 levels and autophagic degradation directly impact the proteasome landscape

2021; Elsevier BV; Volume: 296; Linguagem: Inglês

10.1016/j.jbc.2021.100468

ISSN

1083-351X

Autores

Alicia Burris, Kenrick A. Waite, Zachary Reuter, Samuel Ockerhausen, Jeroen Roelofs,

Tópico(s)

Endoplasmic Reticulum Stress and Disease

Resumo

The proteasome selectively degrades proteins. It consists of a core particle (CP), which contains proteolytic active sites that can associate with different regulators to form various complexes. How these different complexes are regulated and affected by changing physiological conditions, however, remains poorly understood. In this study, we focused on the activator Blm10 and the regulatory particle (RP). In yeast, increased expression of Blm10 outcompeted RP for CP binding, which suggests that controlling the cellular levels of Blm10 can affect the relative amounts of RP-bound CP. While strong overexpression of BLM10 almost eliminated the presence of RP-CP complexes, the phenotypes this should induce were not observed. Our results show this was due to the induction of Blm10-CP autophagy under prolonged growth in YPD. Similarly, under conditions of endogenous BLM10 expression, Blm10 was degraded through autophagy as well. This suggests that reducing the levels of Blm10 allows for more CP-binding surfaces and the formation of RP-CP complexes under nutrient stress. This work provides important insights into maintaining the proteasome landscape and how protein expression levels affect proteasome function. The proteasome selectively degrades proteins. It consists of a core particle (CP), which contains proteolytic active sites that can associate with different regulators to form various complexes. How these different complexes are regulated and affected by changing physiological conditions, however, remains poorly understood. In this study, we focused on the activator Blm10 and the regulatory particle (RP). In yeast, increased expression of Blm10 outcompeted RP for CP binding, which suggests that controlling the cellular levels of Blm10 can affect the relative amounts of RP-bound CP. While strong overexpression of BLM10 almost eliminated the presence of RP-CP complexes, the phenotypes this should induce were not observed. Our results show this was due to the induction of Blm10-CP autophagy under prolonged growth in YPD. Similarly, under conditions of endogenous BLM10 expression, Blm10 was degraded through autophagy as well. This suggests that reducing the levels of Blm10 allows for more CP-binding surfaces and the formation of RP-CP complexes under nutrient stress. This work provides important insights into maintaining the proteasome landscape and how protein expression levels affect proteasome function. Most protein degradation in eukaryotic cells is performed by a large complex known as the proteasome. Unlike lysosomal and secreted proteases, proteasomes sequester proteolytic active sites away from potential substrates as they are located within a barrel-shaped structure known as the core particle (also known as CP or 20S). CP is composed of two sets of 14 unique subunits. These subunits are arranged in four stacked heptameric rings. α subunits 1–7 form the outer rings while the two inner rings are composed of β subunits 1–7. Three pairs of proteolytic β subunits, each with distinct specificity, are responsible for cleaving substrates into short polypeptides (1Groll M. Ditzel L. Lowe J. Stock D. Bochtler M. Bartunik H.D. Huber R. Structure of 20S proteasome from yeast at 2.4 A resolution.Nature. 1997; 386: 463-471Crossref PubMed Scopus (1897) Google Scholar, 2Marques A.J. Palanimurugan R. Matias A.C. Ramos P.C. Dohmen R.J. Catalytic mechanism and assembly of the proteasome.Chem. Rev. 2009; 109: 1509-1536Crossref PubMed Scopus (140) Google Scholar). Peptides released by the proteasome are further processed by cytosolic peptidases to produce intermediates for various metabolic processes (3Reits E. Griekspoor A. Neijssen J. Groothuis T. Jalink K. van Veelen P. Janssen H. Calafat J. Drijfhout J.W. Neefjes J. Peptide diffusion, protection, and degradation in nuclear and cytoplasmic compartments before antigen presentation by MHC class I.Immunity. 2003; 18: 97-108Abstract Full Text Full Text PDF PubMed Scopus (253) Google Scholar, 4Vigneron N. Van den Eynde B.J. Proteasome subtypes and regulators in the processing of antigenic peptides presented by class I molecules of the major histocompatibility complex.Biomolecules. 2014; 4: 994-1025Crossref PubMed Scopus (57) Google Scholar). While the CP alone appears to be able to degrade certain classes of substrates, e.g., under oxidative stress, it is well established that the majority of substrates cannot be degraded by CP alone (5Groll M. Bajorek M. Kohler A. Moroder L. Rubin D.M. Huber R. Glickman M.H. Finley D. A gated channel into the proteasome core particle.Nat. Struct. Biol. 2000; 7: 1062-1067Crossref PubMed Scopus (623) Google Scholar). The reason for this is threefold; first, most substrates are labeled for degradation with a posttranslational modification on lysine residues known as ubiquitination. The receptors that recognize this modification are not part of the CP. Second, most substrates contain stable tertiary and quaternary structures, which require unfolding before they can enter the CP. Third, even when substrates are unfolded or disordered, entry into the catalytic chamber is restricted by a gate composed of the N termini of α subunits (5Groll M. Bajorek M. Kohler A. Moroder L. Rubin D.M. Huber R. Glickman M.H. Finley D. A gated channel into the proteasome core particle.Nat. Struct. Biol. 2000; 7: 1062-1067Crossref PubMed Scopus (623) Google Scholar). The association of CP with the regulatory particle (RP or 19S) eliminates all of these limitations and results in the formation of the 26S proteasome (here used to refer to CP complexes with one or two RPs). The RP is composed of 19 polypeptides. Three of these function as intrinsic ubiquitin receptors, Rpn1, Rpn10, and Rpn13, and thus are able to bind substrates (6Deveraux Q. Ustrell V. Pickart C. Rechsteiner M. A 26 S protease subunit that binds ubiquitin conjugates.J. Biol. Chem. 1994; 269: 7059-7061Abstract Full Text PDF PubMed Google Scholar, 7Husnjak K. Elsasser S. Zhang N.X. Chen X. Randles L. Shi Y. Hofmann K. Walters K.J. Finley D. Dikic I. Proteasome subunit Rpn13 is a novel ubiquitin receptor.Nature. 2008; 453: 481-488Crossref PubMed Scopus (461) Google Scholar, 8Schreiner P. Chen X. Husnjak K. Randles L. Zhang N. Elsasser S. Finley D. Dikic I. Walters K.J. Groll M. Ubiquitin docking at the proteasome through a novel pleckstrin-homology domain interaction.Nature. 2008; 453: 548-552Crossref PubMed Scopus (246) Google Scholar, 9Shi Y. Chen X. Elsasser S. Stocks B.B. Tian G. Lee B.H. Shi Y. Zhang N. de Poot S.A. Tuebing F. Sun S. Vannoy J. Tarasov S.G. Engen J.R. Finley D. et al.Rpn1 provides adjacent receptor sites for substrate binding and deubiquitination by the proteasome.Science. 2016; 351aad9421Crossref PubMed Scopus (151) Google Scholar). These substrates are deubiquitinated by various deubiquitinating enzymes including the intrinsic RP subunit Rpn11 and the proteasome-associated enzyme Ubp6. A hexameric ring of six AAA-ATPases (Rpt1-6) utilizes ATP to unfold substrates and translocate them into the CP for degradation. Several of the ATPase subunits have C-terminal tails with a conserved Hb-Y-X motif (Hb refers to a hydrophobic amino acid, Y is tyrosine, and X can be any amino acid). Docking of these tails into pockets on the surface of the CP α-ring contributes to the affinity between RP and CP as well as induces conformational changes that open the gate and allow for substrate entry into the core particle (10Rabl J. Smith D.M. Yu Y. Chang S.C. Goldberg A.L. Cheng Y. Mechanism of gate opening in the 20S proteasome by the proteasomal ATPases.Mol. Cell. 2008; 30: 360-368Abstract Full Text Full Text PDF PubMed Scopus (273) Google Scholar, 11Sadre-Bazzaz K. Whitby F.G. Robinson H. Formosa T. Hill C.P. Structure of a Blm10 complex reveals common mechanisms for proteasome binding and gate opening.Mol. Cell. 2010; 37: 728-735Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar, 12Smith D.M. Chang S.C. Park S. Finley D. Cheng Y. Goldberg A.L. Docking of the proteasomal ATPases' carboxyl termini in the 20S proteasome's alpha ring opens the gate for substrate entry.Mol. Cell. 2007; 27: 731-744Abstract Full Text Full Text PDF PubMed Scopus (375) Google Scholar). Thus, 26S proteasomes bind, unfold, and subsequently degrade the majority of physiologically important substrates. Besides RP, several other complexes can associate with the same surface of CP that is occupied by RP, namely the 11S activator (REGα-β and REGγ, a.k.a. as PA28αβ and PA28γ; not found in yeast), Pba1-Pba2/PAC1-PAC2, Blm10/PA200, and Fub1/PI31 (11Sadre-Bazzaz K. Whitby F.G. Robinson H. Formosa T. Hill C.P. Structure of a Blm10 complex reveals common mechanisms for proteasome binding and gate opening.Mol. Cell. 2010; 37: 728-735Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar, 13Stadtmueller B.M. Kish-Trier E. Ferrell K. Petersen C.N. Robinson H. Myszka D.G. Eckert D.M. Formosa T. Hill C.P. Structure of a proteasome Pba1-Pba2 complex: Implications for proteasome assembly, activation, and biological function.J. Biol. Chem. 2012; 287: 37371-37382Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar, 14Strehl B. Seifert U. Kruger E. Heink S. Kuckelkorn U. Kloetzel P.M. Interferon-gamma, the functional plasticity of the ubiquitin-proteasome system, and MHC class I antigen processing.Immunol. Rev. 2005; 207: 19-30Crossref PubMed Scopus (175) Google Scholar, 15McCutchen-Maloney S.L. Matsuda K. Shimbara N. Binns D.D. Tanaka K. Slaughter C.A. DeMartino G.N. cDNA cloning, expression, and functional characterization of PI31, a proline-rich inhibitor of the proteasome.J. Biol. Chem. 2000; 275: 18557-18565Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar). However, none of these can hydrolyze ATP or are able to recognize ubiquitinated substrates suggesting that their function is either different or more specialized. In general terms, they could function as a competitor to prevent RP binding. Competitive binding has the potential to negatively regulate proteasome activity, as has been found and proposed for PI31 (15McCutchen-Maloney S.L. Matsuda K. Shimbara N. Binns D.D. Tanaka K. Slaughter C.A. DeMartino G.N. cDNA cloning, expression, and functional characterization of PI31, a proline-rich inhibitor of the proteasome.J. Biol. Chem. 2000; 275: 18557-18565Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar, 16Li X. Thompson D. Kumar B. DeMartino G.N. Molecular and cellular roles of PI31 (PSMF1) protein in regulation of proteasome function.J. Biol. Chem. 2014; 289: 17392-17405Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). A second possible function involves a role during CP assembly or maturation. Pba1-Pba2 was shown to stimulate α-ring assembly and prevented RP from associating with immature CP (17Hirano Y. Hendil K.B. Yashiroda H. Iemura S. Nagane R. Hioki Y. Natsume T. Tanaka K. Murata S. A heterodimeric complex that promotes the assembly of mammalian 20S proteasomes.Nature. 2005; 437: 1381-1385Crossref PubMed Scopus (183) Google Scholar, 18Wani P.S. Rowland M.A. Ondracek A. Deeds E.J. Roelofs J. Maturation of the proteasome core particle induces an affinity switch that controls regulatory particle association.Nat. Commun. 2015; 6: 6384Crossref PubMed Scopus (26) Google Scholar). Another possible function is in regulating the localization or transport of CP, e.g., into or out of the nucleus. Such a role has been proposed for Blm10 in yeast (19Weberruss M.H. Savulescu A.F. Jando J. Bissinger T. Harel A. Glickman M.H. Enenkel C. Blm10 facilitates nuclear import of proteasome core particles.EMBO J. 2013; 32: 2697-2707Crossref PubMed Scopus (34) Google Scholar). Finally, these CP-associated proteins may function as distinct, specialized degradation complexes. Here, the degradation would not depend on ubiquitination-based substrate targeting (considering the lack of ubiquitin receptors) or protein unfolding (considering the lack of ATPase activity in these regulators). This role is consistent with the proposed function for REGγ in degrading intrinsically disordered proteins in the nucleus (20Chen X. Barton L.F. Chi Y. Clurman B.E. Roberts J.M. Ubiquitin-independent degradation of cell-cycle inhibitors by the REGgamma proteasome.Mol. Cell. 2007; 26: 843-852Abstract Full Text Full Text PDF PubMed Scopus (223) Google Scholar, 21Li X. Amazit L. Long W. Lonard D.M. Monaco J.J. O'Malley B.W. Ubiquitin- and ATP-independent proteolytic turnover of p21 by the REGgamma-proteasome pathway.Mol. Cell. 2007; 26: 831-842Abstract Full Text Full Text PDF PubMed Scopus (217) Google Scholar, 22Li X. Lonard D.M. Jung S.Y. Malovannaya A. Feng Q. Qin J. Tsai S.Y. Tsai M.J. O'Malley B.W. The SRC-3/AIB1 coactivator is degraded in a ubiquitin- and ATP-independent manner by the REGgamma proteasome.Cell. 2006; 124: 381-392Abstract Full Text Full Text PDF PubMed Scopus (219) Google Scholar, 23Nie J. Wu M. Wang J. Xing G. He F. Zhang L. REGgamma proteasome mediates degradation of the ubiquitin ligase Smurf1.FEBS Lett. 2010; 584: 3021-3027Crossref PubMed Scopus (26) Google Scholar, 24Suzuki R. Moriishi K. Fukuda K. Shirakura M. Ishii K. Shoji I. Wakita T. Miyamura T. Matsuura Y. Suzuki T. Proteasomal turnover of hepatitis C virus core protein is regulated by two distinct mechanisms: A ubiquitin-dependent mechanism and a ubiquitin-independent but PA28gamma-dependent mechanism.J. Virol. 2009; 83: 2389-2392Crossref PubMed Scopus (46) Google Scholar). The above models act under the assumption that CP forms homogeneous complexes where one type of activator binds both ends of the CP; however, CP can actually form hybrid complexes where RP binds to one end, while the other end is occupied by one of these alternative regulators. Such complexes have been purified from cells and detected in cell lysates (25Hendil K.B. Khan S. Tanaka K. Simultaneous binding of PA28 and PA700 activators to 20 S proteasomes.Biochem. J. 1998; 332: 749-754Crossref PubMed Scopus (190) Google Scholar, 26Hoffman L. Pratt G. Rechsteiner M. Multiple forms of the 20 S multicatalytic and the 26 S ubiquitin/ATP-dependent proteases from rabbit reticulocyte lysate.J. Biol. Chem. 1992; 267: 22362-22368Abstract Full Text PDF PubMed Google Scholar, 27Tanahashi N. Murakami Y. Minami Y. Shimbara N. Hendil K.B. Tanaka K. Hybrid proteasomes. Induction by interferon-gamma and contribution to ATP-dependent proteolysis.J. Biol. Chem. 2000; 275: 14336-14345Abstract Full Text Full Text PDF PubMed Scopus (314) Google Scholar). The formation of hybrid complexes could cause a change in cellular localization of RP-CP complexes. Binding of other activators may also induce allosteric changes inside the CP that affect the specificity of proteolytic cleavage or change cleavage dynamics and peptide retention in the catalytic chamber. As such, hybrid complexes could degrade ubiquitinated proteins, but produce peptides of different composition and length as final products that are released by the proteasome. REGα-β forms heptameric rings that stimulate gate opening through a mechanism different from RP. The C-terminal Hb-Y-X motifs of REGα-β contribute to CP binding but are not involved in gate opening. Instead, gate opening occurs through an activation loop within REGα-β that induces conformational changes required for access (28Forster A. Masters E.I. Whitby F.G. Robinson H. Hill C.P. The 1.9 A structure of a proteasome-11S activator complex and implications for proteasome-PAN/PA700 interactions.Mol. Cell. 2005; 18: 589-599Abstract Full Text Full Text PDF PubMed Scopus (187) Google Scholar, 29Zhang Z. Clawson A. Realini C. Jensen C.C. Knowlton J.R. Hill C.P. Rechsteiner M. Identification of an activation region in the proteasome activator REGalpha.Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 2807-2811Crossref PubMed Scopus (77) Google Scholar). Blm10 is a unique regulator as it has been observed bound to both mature and immature CP. Blm10 can be bound to CP alone or in hybrid complexes with RP. Blm10 in Saccharomyces cerevisiae (PA200 in humans) is a single ∼240 kDa polypeptide that contains multiple heat repeats, a bromodomain-like region, and an Hb-Y-X motif at its C terminus. Similar to other activators, this motif allows Blm10 to bind to a pocket on the surface of the α ring, specifically the α5-α6 pocket (11Sadre-Bazzaz K. Whitby F.G. Robinson H. Formosa T. Hill C.P. Structure of a Blm10 complex reveals common mechanisms for proteasome binding and gate opening.Mol. Cell. 2010; 37: 728-735Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar). However, unlike other regulators, Blm10 binds proteasomes as a monomeric protein. Binding of Blm10 to the CP induces partial gate opening and increases peptidase activity; therefore, Blm10 has been described as an activator (30Dange T. Smith D. Noy T. Rommel P.C. Jurzitza L. Cordero R.J. Legendre A. Finley D. Goldberg A.L. Schmidt M. Blm10 protein promotes proteasomal substrate turnover by an active gating mechanism.J. Biol. Chem. 2011; 286: 42830-42839Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). Indeed, Blm10-CP complexes have been reported to be involved in degradation of short peptides and unstructured proteins such as tau, which is reasonable considering those do not require ubiquitination or ATPase-dependent unfolding (30Dange T. Smith D. Noy T. Rommel P.C. Jurzitza L. Cordero R.J. Legendre A. Finley D. Goldberg A.L. Schmidt M. Blm10 protein promotes proteasomal substrate turnover by an active gating mechanism.J. Biol. Chem. 2011; 286: 42830-42839Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). In addition, Blm10 appears to be required for the degradation of Sfp1 and histones, further supporting a role for Blm10 in protein degradation (31Lopez A.D. Tar K. Krugel U. Dange T. Ros I.G. Schmidt M. Proteasomal degradation of Sfp1 contributes to the repression of ribosome biogenesis during starvation and is mediated by the proteasome activator Blm10.Mol. Biol. Cell. 2011; 22: 528-540Crossref PubMed Scopus (32) Google Scholar, 32Mandemaker I.K. Geijer M.E. Kik I. Bezstarosti K. Rijkers E. Raams A. Janssens R.C. Lans H. Hoeijmakers J.H. Demmers J.A. Vermeulen W. Marteijn J.A. DNA damage-induced replication stress results in PA200-proteasome-mediated degradation of acetylated histones.EMBO Rep. 2018; 19Crossref Scopus (26) Google Scholar, 33Qian M.X. Pang Y. Liu C.H. Haratake K. Du B.Y. Ji D.Y. Wang G.F. Zhu Q.Q. Song W. Yu Y. Zhang X.X. Huang H.T. Miao S. Chen L.B. Zhang Z.H. et al.Acetylation-mediated proteasomal degradation of core histones during DNA repair and spermatogenesis.Cell. 2013; 153: 1012-1024Abstract Full Text Full Text PDF PubMed Scopus (190) Google Scholar). The presumably folded nature of these substrates might indicate a role for hybrid RP-CP-Blm10 complexes. However, a clear mechanism of action for these hybrid complexes is unknown and other factors might assist in the degradation. Considering that Blm10 is also found on an immature form of CP, a role in CP maturation has also been proposed. However, its function here seems very different from the assembly chaperones Pba1-Pba2, which seems to exclusively bind to immature CP. Pba1-Pba 2 has a very low affinity for mature CP and a high affinity for the immature form (18Wani P.S. Rowland M.A. Ondracek A. Deeds E.J. Roelofs J. Maturation of the proteasome core particle induces an affinity switch that controls regulatory particle association.Nat. Commun. 2015; 6: 6384Crossref PubMed Scopus (26) Google Scholar). Consistent with this, Pba1-Pba2 is more embedded in the α ring of immature CP compared with mature CP, thus restricting RP-CP interactions (34Kock M. Nunes M.M. Hemann M. Kube S. Dohmen R.J. Herzog F. Ramos P.C. Wendler P. Proteasome assembly from 15S precursors involves major conformational changes and recycling of the Pba1-Pba2 chaperone.Nat. Commun. 2015; 6: 6123Crossref PubMed Scopus (33) Google Scholar). While Blm10 could potentially perform a similar role for immature CP, there is no apparent difference in Blm10 affinity for mature versus immature CP that would allow for an exchange of regulators in maturation. Consistent with this, deletion of Blm10 showed no obvious defect in CP maturation (35Fehlker M. Wendler P. Lehmann A. Enenkel C. Blm3 is part of nascent proteasomes and is involved in a late stage of nuclear proteasome assembly.EMBO Rep. 2003; 4: 959-963Crossref PubMed Scopus (96) Google Scholar, 36Marques A.J. Glanemann C. Ramos P.C. Dohmen R.J. The C-terminal extension of the beta7 subunit and activator complexes stabilize nascent 20 S proteasomes and promote their maturation.J. Biol. Chem. 2007; 282: 34869-34876Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar). Over the years, many other roles for Blm10 and PA200 have been suggested, including involvement in processes such as spermatogenesis, DNA repair, histone degradation, CP sequestration into proteasome storage granules (PSGs), and degradation of mitochondrial proteins (19Weberruss M.H. Savulescu A.F. Jando J. Bissinger T. Harel A. Glickman M.H. Enenkel C. Blm10 facilitates nuclear import of proteasome core particles.EMBO J. 2013; 32: 2697-2707Crossref PubMed Scopus (34) Google Scholar, 32Mandemaker I.K. Geijer M.E. Kik I. Bezstarosti K. Rijkers E. Raams A. Janssens R.C. Lans H. Hoeijmakers J.H. Demmers J.A. Vermeulen W. Marteijn J.A. DNA damage-induced replication stress results in PA200-proteasome-mediated degradation of acetylated histones.EMBO Rep. 2018; 19Crossref Scopus (26) Google Scholar, 33Qian M.X. Pang Y. Liu C.H. Haratake K. Du B.Y. Ji D.Y. Wang G.F. Zhu Q.Q. Song W. Yu Y. Zhang X.X. Huang H.T. Miao S. Chen L.B. Zhang Z.H. et al.Acetylation-mediated proteasomal degradation of core histones during DNA repair and spermatogenesis.Cell. 2013; 153: 1012-1024Abstract Full Text Full Text PDF PubMed Scopus (190) Google Scholar, 35Fehlker M. Wendler P. Lehmann A. Enenkel C. Blm3 is part of nascent proteasomes and is involved in a late stage of nuclear proteasome assembly.EMBO Rep. 2003; 4: 959-963Crossref PubMed Scopus (96) Google Scholar, 36Marques A.J. Glanemann C. Ramos P.C. Dohmen R.J. The C-terminal extension of the beta7 subunit and activator complexes stabilize nascent 20 S proteasomes and promote their maturation.J. Biol. Chem. 2007; 282: 34869-34876Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar, 37Blickwedehl J. Agarwal M. Seong C. Pandita R.K. Melendy T. Sung P. Pandita T.K. Bangia N. Role for proteasome activator PA200 and postglutamyl proteasome activity in genomic stability.Proc. Natl. Acad. Sci. U. S. A. 2008; 105: 16165-16170Crossref PubMed Scopus (77) Google Scholar, 38Blickwedehl J. McEvoy S. Wong I. Kousis P. Clements J. Elliott R. Cresswell P. Liang P. Bangia N. Proteasomes and proteasome activator 200 kDa (PA200) accumulate on chromatin in response to ionizing radiation.Radiat. Res. 2007; 167: 663-674Crossref PubMed Scopus (41) Google Scholar, 39Khor B. Bredemeyer A.L. Huang C.Y. Turnbull I.R. Evans R. Maggi Jr., L.B. White J.M. Walker L.M. Carnes K. Hess R.A. Sleckman B.P. Proteasome activator PA200 is required for normal spermatogenesis.Mol. Cell Biol. 2006; 26: 2999-3007Crossref PubMed Scopus (104) Google Scholar, 40Tar K. Dange T. Yang C. Yao Y. Bulteau A.L. Salcedo E.F. Braigen S. Bouillaud F. Finley D. Schmidt M. Proteasomes associated with the Blm10 activator protein antagonize mitochondrial fission through degradation of the fission protein Dnm1.J. Biol. Chem. 2014; 289: 12145-12156Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar, 41Ustrell V. Hoffman L. Pratt G. Rechsteiner M. PA200, a nuclear proteasome activator involved in DNA repair.EMBO J. 2002; 21: 3516-3525Crossref PubMed Scopus (254) Google Scholar). The predicted bromodomain-like region of Blm10/PA200 was shown to specifically bind acetylated histones, which led to the subsequent degradation of histones by PA200-CP complexes (32Mandemaker I.K. Geijer M.E. Kik I. Bezstarosti K. Rijkers E. Raams A. Janssens R.C. Lans H. Hoeijmakers J.H. Demmers J.A. Vermeulen W. Marteijn J.A. DNA damage-induced replication stress results in PA200-proteasome-mediated degradation of acetylated histones.EMBO Rep. 2018; 19Crossref Scopus (26) Google Scholar, 33Qian M.X. Pang Y. Liu C.H. Haratake K. Du B.Y. Ji D.Y. Wang G.F. Zhu Q.Q. Song W. Yu Y. Zhang X.X. Huang H.T. Miao S. Chen L.B. Zhang Z.H. et al.Acetylation-mediated proteasomal degradation of core histones during DNA repair and spermatogenesis.Cell. 2013; 153: 1012-1024Abstract Full Text Full Text PDF PubMed Scopus (190) Google Scholar). However, recent structural work on the PA200-CP complex raises some questions in this regard and shows that PA200 binds to inositol phosphates, which have been shown to bind and regulate histone deacetylases (42Toste Rego A. da Fonseca P.C.A. Characterization of fully recombinant human 20S and 20S-PA200 proteasome complexes.Mol. Cell. 2019; 76: 138-147.e135Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar, 43Guan H. Wang Y. Yu T. Huang Y. Li M. Saeed A. Perculija V. Li D. Xiao J. Wang D. Zhu P. Ouyang S. Cryo-EM structures of the human PA200 and PA200-20S complex reveal regulation of proteasome gate opening and two PA200 apertures.PLoS Biol. 2020; 18e3000654Crossref PubMed Scopus (11) Google Scholar). Furthermore, binding of PA200 results in an unusually wide α ring. These conformational changes are propagated to the active site β subunits, thereby changing the structure of the active sites and affecting proteasome activity. Specifically, activation of β2 trypsin-like activity was observed along with a slight inhibition of β5 and β1 activity (44Goldstein A.L. McCusker J.H. Three new dominant drug resistance cassettes for gene disruption in Saccharomyces cerevisiae.Yeast. 1999; 15: 1541-1553Crossref PubMed Scopus (1307) Google Scholar). In order to gain insight into the physiological role of Blm10 containing complexes, we altered the abundance of this protein in yeast and monitored the effects of its overexpression on the proteasome landscape. Further, we sought to determine how Blm10-bound complexes are affected by starvation conditions. The proteasome CP is a cylindrically shaped structure able to bind one of numerous regulators. However, binding of these regulators is mutually exclusive as they bind the same interface of CP. Thus, there are a variety of proteasome complexes that contain one or two copies of a particular regulator. Additionally, hybrid complexes (CP with two different regulators) can form. In all, there is the potential for a varied landscape of proteasome complexes. Several of these potential complexes have been observed in yeast as well as mammalian cells, e.g. (45Cascio P. Call M. Petre B.M. Walz T. Goldberg A.L. Properties of the hybrid form of the 26S proteasome containing both 19S and PA28 complexes.EMBO J. 2002; 21: 2636-2645Crossref PubMed Scopus (173) Google Scholar, 46Kopp F. Dahlmann B. Kuehn L. Reconstitution of hybrid proteasomes from purified PA700-20 S complexes and PA28alphabeta activator: Ultrastructure and peptidase activities.J. Mol. Biol. 2001; 313: 465-471Crossref PubMed Scopus (61) Google Scholar); however, our understanding of the mechanisms and regulations that govern this landscape is limited. In lysates of a strain where the endogenous copy of α1 was tagged with GFP, a number of these complexes can be distinguished on native gel (Fig. 1A, lane 1). Whole-cell lysate prepared by cryo-grinding was separated on native gel and imaged for GFP to identify the native complexes that contained the CP subunit α1. A number of species can be readily identified; from top to bottom these are: CP with RP bound on both ends (RP2-CP), CP-RP with Blm10 bound (Blm10-CP-RP), CP with one RP (RP-CP), CP with Blm10 bound on both ends (Blm102-CP), CP with Blm10 bound on one end, and free CP. Assignment of these species is based on extensive work by us and other laboratories (47De La Mota-Peynado A. Lee S.Y. Pierce B.M. Wani P. Singh C.R. Roelofs J. The proteasome-associated protein Ecm29 inhibits proteasomal ATPase activity and in vivo protein degradation by the proteasome.J. Biol. Chem. 2013; 288: 29467-29481Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar, 48Leggett D.S. Hanna J. Borodovsky A. Crosas B. Schmidt M. Baker R.T. Walz T. Ploegh H. Finley D. Multiple associated proteins regulate proteasome structure and function.Mol. Cell. 2002; 10: 495-507Abstract Full Text Full Text PDF PubMed Scopus (504) Google Scholar, 49Schmidt M. Haas W. Crosas B. Santamaria P.G. Gygi S.P. Walz T. Finley D. The HEAT repeat protein Blm10 regulates the yeast proteasome by capping the core particle.Nat. Struct. Mol. Biol. 2005; 12: 294-303Crossref PubMed Scopus (146) Google Scholar). It should be noted that the Blm10-CP-RP band could contain Ecm29, as this proteasome-associated 210 kDa protein can bind to RP-CP, Blm10-CP-RP, and RP2-CP complexes. Further, Ecm29 retards the migration of complexes on gel when it is bound (47De La Mota-Peynado A. Lee S.Y. Pierce B.M. Wani P. Singh C.R. Roelofs J. The proteasome-associated protein Ecm29 inhibits proteasomal ATPase activity and in vivo protein degradation by the proteasome.J. Biol. Chem. 2013; 288: 29467-29481Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar, 48Leggett D.S. Hanna J. Borodovsky A. Crosas B. Schmidt M. Baker R.T. Walz T. Ploegh H. Finley D. Multiple associated proteins regulate proteasome structure and function.Mol. Cell. 2002; 10: 495-507Abstract Full Text Full Text PDF PubMed Scopus (504) Google Scholar, 50Kleijnen M.F. Roelofs J. Park S. Hathaway N.A. Glickman M. King R.W. Finley D. Stability of the proteasome can be regulated allosterically through engagement of its proteolytic active sites.Nat. Struct. Mol. Biol. 2007; 14: 1180-1188Crossref PubMed Scopus (117) Google Scholar, 51Lee S.Y.

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