Localization of the regulatory particle subunit Sem1 in the 26S proteasome

Bohn, Stefan; Sakata, Eri; Beck, Florian; Pathare, Ganesh Ramnath; Schnitger, Jérôme; Nagy, István; Baumeister, Wolfgang; Förster, Friedrich

doi:10.1016/j.bbrc.2013.04.069

Cited by 28 publications

(35 citation statements)

References 33 publications

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“…In comparison to these reports, it is noted that our DSSO XL-MS strategy was able to determine two additional interactions between the small Rpn15/Sem1 subunit to Rpn3 and Rpn7 respectively, which were not detected by other approaches (47,48). This finding was later confirmed by a more targeted approach through EM difference mapping between wild-type Rpn15 and an Rpn15 deletion strain (9). This further indicates that XL-MS analysis can provide complementary information to EM-based structural analysis, and combinatory approaches integrating various technologies are beneficial in the structural characterization of heterogeneous and dynamic protein complexes such as proteasomes.…”

Section: Fig 4 Ms Sequencing Of Inter-linked Peptides (A)supporting

confidence: 60%

“…In comparison, Rpn1 is conformationally variable and positioned at the periphery of the ATPase ring (48). Moreover, the two ubiquitin receptors (Rpn10 and Rpn13) and a deubiquitinase (Rpn11) appear to be in a forked arrangement with Rpn11 in the center bottom and the two receptors in the top corners (9,48). This suggests an arrangement where the polyUb chain of a protein substrate is bound to the distal receptors, and the base of the chain is exposed to the deubiquitinase.…”

Section: Fig 4 Ms Sequencing Of Inter-linked Peptides (A)mentioning

confidence: 98%

“…Unlike the 20S CP, which has a highly conserved and ordered structure, the 19S RP is less ordered, highly dynamic, and heterogeneous in nature and, thus, has proved to be much more difficult to characterize by traditional techniques such as X-ray crystallography and nuclear magnetic resonance. Therefore, alternative strategies based on low-resolution structural tools, such as cryo-electron microscopy (cryoEM) and crosslinking mass spectrometry (XL-MS), have been developed and utilized to elucidate structures of the 19S RP and 26S holocomplex (4,8,9,17,26,42,47,48,58,63,73). These studies have provided new insights on the structural framework of the 26S proteasome, thus significantly improving our understanding of proteasome function (56).…”

Section: Structural Characterization Of the 26s Proteasome Complexmentioning

confidence: 99%

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Characterizing the Dynamics of Proteasome Complexes by Proteomics Approaches

Kaake

Kao²,

Yu³

et al. 2014

Antioxidants & Redox Signaling

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Section: Fig 4 Ms Sequencing Of Inter-linked Peptides (A)supporting

confidence: 60%

Section: Fig 4 Ms Sequencing Of Inter-linked Peptides (A)mentioning

confidence: 98%

Section: Structural Characterization Of the 26s Proteasome Complexmentioning

confidence: 99%

See 1 more Smart Citation

Characterizing the Dynamics of Proteasome Complexes by Proteomics Approaches

Kaake

Kao²,

Yu³

et al. 2014

Antioxidants & Redox Signaling

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“…The small lid subunit Rpn15/Dss1, which was recently suggested to function as a ubiquitin receptor (38), is positioned between Rpn7 and Rpn3 (Fig. 4E) (14,39). The lid shields the Rpn8/11 heterodimer, which projects the active site of Rpn11 near the mouth of the AAA module.…”

Section: Resultsmentioning

confidence: 95%

Structure of the human 26S proteasome at a resolution of 3.9 Å

Schweitzer

Aufderheide

Rudack

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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Protein degradation in eukaryotic cells is performed by the UbiquitinProteasome System (UPS). The 26S proteasome holocomplex consists of a core particle (CP) that proteolytically degrades polyubiquitylated proteins, and a regulatory particle (RP) containing the AAA-ATPase module. This module controls access to the proteolytic chamber inside the CP and is surrounded by non-ATPase subunits (Rpns) that recognize substrates and deubiquitylate them before unfolding and degradation. The architecture of the 26S holocomplex is highly conserved between yeast and humans. The structure of the human 26S holocomplex described here reveals previously unidentified features of the AAA-ATPase heterohexamer. One subunit, Rpt6, has ADP bound, whereas the other five have ATP in their binding pockets. Rpt6 is structurally distinct from the other five Rpt subunits, most notably in its pore loop region. For Rpns, the map reveals two main, previously undetected, features: the C terminus of Rpn3 protrudes into the mouth of the ATPase ring; and Rpn1 and Rpn2, the largest proteasome subunits, are linked by an extended connection. The structural features of the 26S proteasome observed in this study are likely to be important for coordinating the proteasomal subunits during substrate processing.proteostasis | cryo-electron microscopy | AAA-ATPase | integrative modeling T he 26S proteasome is an ATP-dependent multisubunit protease degrading polyubiquitylated proteins (1, 2). It operates at the executive end of the Ubiquitin-Proteasome System (UPS) and has a key role in cellular proteostasis. The 26S proteasome selectively removes misfolded proteins or proteins no longer needed and it is critically involved in numerous cellular processes such as protein quality control, regulation of metabolism, cell cycle control, or antigen presentation. Malfunctions of the UPS are associated with various pathologies, including neurodegenerative diseases and cancer. Therefore, the proteasome is an important pharmaceutical target, and a high-resolution structure is a prerequisite for structure-based drug design (3).The 26S proteasome comprises the 20S cylindrical core particle (CP), where proteolysis takes place, and 19S regulatory particles (RPs). In cellular environments, 26S holocomplexes with either one or two RPs bound to the ends of the cylindershaped CP coexist (4). The role of the RPs is to recruit ubiquitylated substrates, to cleave off their polyubiquitin tags, and to unfold and translocate them into the CP for degradation into short peptides. Whereas X-ray crystallography has revealed the atomic structures first of archaeal 20S proteasome (5) and subsequently of the yeast (6) and mammalian proteasome (7), only lower-resolution structures were available for the 26S holocomplex. Given the compositional and conformational heterogeneity of the RP, single-particle cryo-electron microscopy (cryo-EM) has been the most successful approach to determining the structure of the 26S holocomplex (8). At this point, the most detailed insights have been obtained ...

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“…We also incorporated recent high-resolution structural data for several components: atomic structures of the dimer of the Rpn8/Rpn11 Mpr1-Pad1 N-terminal (MPN) domains (22), a major portion of Rpn12 (23) and Csn1 (24), a paralog of Rpn7, a computational model of the helical lid bundle (25), and the location of the C-terminal segment Sem1 in the 26S holocomplex (26).…”

Section: Distinct 26s Proteasome Conformations At Subnanometer Resolumentioning

confidence: 99%

Deep classification of a large cryo-EM dataset defines the conformational landscape of the 26S proteasome

Unverdorben

Beck

Śledź

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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179

289

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The 26S proteasome is a 2.5 MDa molecular machine that executes the degradation of substrates of the ubiquitin-proteasome pathway. The molecular architecture of the 26S proteasome was recently established by cryo-EM approaches. For a detailed understanding of the sequence of events from the initial binding of polyubiquitylated substrates to the translocation into the proteolytic core complex, it is necessary to move beyond static structures and characterize the conformational landscape of the 26S proteasome. To this end we have subjected a large cryo-EM dataset acquired in the presence of ATP and ATP-γS to a deep classification procedure, which deconvolutes coexisting conformational states. Highly variable regions, such as the density assigned to the largest subunit, Rpn1, are now well resolved and rendered interpretable. Our analysis reveals the existence of three major conformations: in addition to the previously described ATP-hydrolyzing (ATP h ) and ATP-γS conformations, an intermediate state has been found. Its AAA-ATPase module adopts essentially the same topology that is observed in the ATP h conformation, whereas the lid is more similar to the ATP-γS bound state. Based on the conformational ensemble of the 26S proteasome in solution, we propose a mechanistic model for substrate recognition, commitment, deubiquitylation, and translocation into the core particle.conformational switching | proteolysis | proteostasis | quality control I n the ubiquitin-proteasome pathway (UPP) the 26S proteasome performs the degradation of intracellular proteins marked for destruction by the covalent attachment of polyubiquitin chains (1-5). The 2.5 MDa complex consists of the barrel-shaped 20S core particle (CP) as well as one or two copies of the 19S regulatory particle (RP) controlling the entry of substrates into the proteolytic chamber of the CP. The structure of the CP was solved by X-ray crystallography a long time ago (6, 7); whereas, the molecular architecture of the 26S holocomplex was determined only recently using cryo-EM single-particle analysis (SPA) approaches (8-10). The RP comprises a ringshaped AAA-ATPase heterohexamer (Rpt1-6) responsible for substrate unfolding and translocation across the CP gate and 13 RP non-ATPases (Rpn1-3, 5-13, 15) surrounding the AAAATPase module. The role of the Rpns is the acceptance of substrates and their deubiquitylation. For a full mechanistic understanding of the early steps of substrate processing it is essential to reveal its dynamics.The compositional and conformational heterogeneity of 26S proteasome preparations makes the structural characterization of this molecular machine challenging (11). Compositional heterogeneity results from multiple proteins that interact with the 26S proteasome substoichiometrically, such as deubiquitylating enzymes (DUBs) or shuttling ubiquitin (Ub) receptors. Conformational switching of the 26S proteasome is mostly driven by ATP binding and hydrolysis. Each of the six distinct Rpt subunits is able to bind and hydrolyze ATP (12-14), which may...

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Localization of the regulatory particle subunit Sem1 in the 26S proteasome

Cited by 28 publications

References 33 publications

Characterizing the Dynamics of Proteasome Complexes by Proteomics Approaches

Characterizing the Dynamics of Proteasome Complexes by Proteomics Approaches

Structure of the human 26S proteasome at a resolution of 3.9 Å

Deep classification of a large cryo-EM dataset defines the conformational landscape of the 26S proteasome

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