Structural characterization of the interaction of Ubp6 with the 26S proteasome

Aufderheide, Antje; Beck, Florian; Stengel, Florian; Hartwig, Michaela; Schweitzer, A.; Pfeifer, Günter; Goldberg, Alfred L.; Sakata, Eri; Baumeister, Wolfgang; Förster, Friedrich

doi:10.1073/pnas.1510449112

Cited by 100 publications

(144 citation statements)

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“…Indeed, we observed an additional density between Rpn1 and the OB ring in the s4 state (Fig. 2B, box), which coincides with the position of Ubp6 (30). Further focused classification of the s4 dataset revealed that ∼50% of the particles do not possess Ubp6 (Fig.…”

Section: Resultsmentioning

confidence: 57%

Structural insights into the functional cycle of the ATPase module of the 26S proteasome

Wehmer

Rudack

Beck

et al. 2017

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

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In eukaryotic cells, the ubiquitin-proteasome system (UPS) is responsible for the regulated degradation of intracellular proteins. The 26S holocomplex comprises the core particle (CP), where proteolysis takes place, and one or two regulatory particles (RPs). The base of the RP is formed by a heterohexameric AAA + ATPase module, which unfolds and translocates substrates into the CP. Applying single-particle cryo-electron microscopy (cryo-EM) and image classification to samples in the presence of different nucleotides and nucleotide analogs, we were able to observe four distinct conformational states (s1 to s4). The resolution of the four conformers allowed for the construction of atomic models of the AAA + ATPase module as it progresses through the functional cycle. In a hitherto unobserved state (s4), the gate controlling access to the CP is open. The structures described in this study allow us to put forward a model for the 26S functional cycle driven by ATP hydrolysis.26S proteasome | cryo-electron microscopy | AAA + ATPase | integrative modeling | single-particle analysis I n eukaryotic cells, the ubiquitin-proteasome system (UPS) degrades proteins that are misfolded, damaged, or no longer needed (1). The 26S proteasome is a 2.5-MDa multisubunit complex comprising the barrel-shaped 20S core particle (CP), where degradation takes place, and one or two 19S regulatory particles (RPs), which bind to the ends of the CP (2-4). The CP is built of four coaxially stacked heteroheptameric rings of α-and β-subunits in the order of αββα (5). Three of the seven β-subunits are catalytically active; substrates are sequestered from the cellular environment in a chamber formed by the two β-rings (6, 7). This self-compartmentalization is a hallmark of many intracellular proteases (8). Substrate access to the proteolytic chamber is controlled by the α-subunit N-terminal extensions, forming a gate (3). Most of proteasome activators, including the RP, contain C-terminal hydrophobic-tyrosine-X (HbYX) motifs, which have been reported to insert into α-ring pockets, triggering gate opening (9-11).The RP is composed of at least 19 canonical subunits and interacts substoichiometrically with an array of proteasomeinteracting proteins that modulate RP function (3). The RP is divided into the "base" and the "lid" subcomplexes. The core of the base is formed by a heterohexameric ATPase associated with various cellular activities (AAA + ATPase), which is the driver of large-scale conformational dynamics of the RP. The AAA + ATPase prepares substrates for degradation in coordination with at least three ubiquitin receptors [26S proteasome non-ATPase regulatory subunit 1 (Rpn1), Rpn10, and Rpn13] (12-14) and a deubiquitylating subunit (Rpn11) (15, 16). Other subunits have structural roles, such as holding the CP and RP together, or in coordinating the movements needed to position the substrates above the pore of the AAA + ATPase for unfolding and translocation (17, 18). The AAA + ATPase is lined by aromatic-hydrophobic loops (pore-1 loops), wh...

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Section: Resultsmentioning

confidence: 57%

Structural insights into the functional cycle of the ATPase module of the 26S proteasome

Wehmer

Rudack

Beck

et al. 2017

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

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“…In contrast to intrinsic proteasome deubiquitinase Rpn11 which promotes degradation, Uch37 and Usp14/Ubp6 appear to antagonize the degradation of ubiquitinated substrates (67,69), suggesting that they are important for recycling ubiquitin and maintaining ubiquitin levels in cells. Although Uch37 interacts with the proteasome through Rpn13/ADRM1 (65,70,71), Usp14/Ubp6 physically binds to proteasome subunit Rpn1 (30,60,72), and interacts with Rpt1 when conjugated to a ubiquitin aldehyde (73,74). The presence of Uch37 and Usp14/Ubp6 in UbR-proteasome complexes further indicate that these deubiquitinases are major players in regulating substrate processing at the proteasome.…”

Section: Discussionmentioning

confidence: 98%

Characterization of Dynamic UbR-Proteasome Subcomplexes by In vivo Cross-linking (X) Assisted Bimolecular Tandem Affinity Purification (XBAP) and Label-free Quantitation

Yang

Wang

et al. 2016

Molecular & Cellular Proteomics

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Proteasomes are protein degradation machines that exist in cells as heterogeneous and dynamic populations. A group of proteins function as ubiquitin receptors (UbRs) that can recognize and deliver ubiquitinated substrates to proteasome complexes for degradation. Defining composition of proteasome complexes engaged with UbRs is critical to understand proteasome function. However, because of the dynamic nature of UbR interactions with the proteasome, it remains technically challenging to capture and isolate UbR-proteasome subcomplexes using conventional purification strategies. As a result, distinguishing the molecular differences among these subcomplexes remains elusive. We have developed a novel affinity purification strategy, in vivo cross-linking (X) assisted bimolecular tandem affinity purification strategy (XBAP), to effectively isolate dynamic UbR-proteasome subcomplexes and define their subunit compositions using label-free quantitative mass spectrometry. In this work, we have analyzed seven distinctive UbR-proteasome complexes and found that all of them contain the same type of the 26S holocomplex. However, selected UbRs interact with a group of proteasome interacting proteins that may link each UbR to specific cellular pathways. The compositional similarities and differences among the seven UbRproteasome subcomplexes have provided new insights on functional entities of proteasomal degradation machineries. The strategy described here represents a gen- Proteasomes are multisubunit protein complexes that are responsible for the degradation of ubiquitinated substrates to maintain cell viability and homeostasis. The 26S proteasome is composed of at least 33 subunits (1-3), which can be divided into two subcomplexes: the 20S catalytic core particle (CP) 1 and the 19S regulatory particle (RP). The 20S CP is responsible for various proteolytic activities, and has a highly conserved "barrel"-like structure consisting of two copies each of 14 nonidentical subunits (␣1-7, ␤1-7), which are arranged into four heptameric rings stacked in the order of ␣ 7 ␤ 7 ␤ 7 ␣ 7 (4, 5). The 19S RP intimately interacts with the 20S CP, regulating its activity. In addition, the 19S RP carries diverse functions including substrate recognition and deubiquitination, protein unfolding, and substrate translocation to the 20S CP for degradation (2, 3, 6 -8). In contrast to the highly ordered and stable structure of the 20S CP, the 19S RP appears to be much more flexible and dynamic (3, 9 -11). Current structural analyses have revealed that six Rpt subunits of the 19S RP form a hexameric AAA-ATPase ring to associate with the cylinder ends of the 20S CP, and are surrounded by a shell of Rpn subunits (9 -11). Apart from the

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“…Improvement of cryo-EM technologies has allowed structural determination of the proteasome at varying resolutions (8,(12)(13)(14)(15)(16)(17)(18). Eight Rpn subunits were identified in the lid by cryo-EM analysis of the proteasome from Saccharomyces cerevisiae (S. cerevisiae) (8) and Schizosaccharomyces pombe (S. pombe) (12).…”

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confidence: 99%

Structure of an endogenous yeast 26S proteasome reveals two major conformational states

Luan

Huang

et al. 2016

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

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The eukaryotic proteasome mediates degradation of polyubiquitinated proteins. Here we report the single-particle cryoelectron microscopy (cryo-EM) structures of the endogenous 26S proteasome from Saccharomyces cerevisiae at 4.6-to 6.3-Å resolution. The fine features of the cryo-EM maps allow modeling of 18 subunits in the regulatory particle and 28 in the core particle. The proteasome exhibits two distinct conformational states, designated M1 and M2, which correspond to those reported previously for the proteasome purified in the presence of ATP-γS and ATP, respectively. These conformations also correspond to those of the proteasome in the presence and absence of exogenous substrate. Structure-guided biochemical analysis reveals enhanced deubiquitylating enzyme activity of Rpn11 upon assembly of the lid. Our structures serve as a molecular basis for mechanistic understanding of proteasome function.protein degradation | proteasome | cryo-EM | structure T he eukaryotic ubiquitin-proteasome system is responsible for the degradation of polyubiquitinated proteins (1). The 26S proteasome consists of one 20S core particle (CP) and two 19S regulatory particles (RPs). The RP is divided into the lid and base assembly intermediates (1). The lid comprises nine Rpn subunits in yeast (Rpn3/5/6/7/8/9/11/12/15) and the base comprises three Rpn subunits (Rpn1/2/13) and six ATPases (Rpt1-6). Rpn10, which consists of an N-terminal von Willebrand factor A (VWA) domain and multiple C-terminal ubiquitin-interacting motifs (UIM), connects the lid and the base. Polyubiquitin (poly-Ub) chains from substrate are recognized by the RP, leading to unfolding of the substrate and its translocation into the CP, where it is degraded.The main function of the lid is to remove poly-Ub chains from the substrate (1). The released Ub chains are recycled via further cleavage into Ub monomers. Six of the nine Rpn subunits in the lid (Rpn3/5/6/7/9/12) contain a solenoid fold followed by a proteasome-CSN-eIF3 (PCI) domain of varying lengths; for Rpn8 and Rpn11, each has an Mpr1-Pad1-N-terminal (MPN) domain. Among all Rpn subunits, Rpn11 is the only deubiquitylating enzyme (DUB); it cleaves the isopeptide bond between the carboxyl terminus of Ub and the e-amino group of Lys in the substrate (2, 3). Except for Rpn15/ Sem1/Dss1, the C-terminal sequences of the other eight Rpn subunits in the lid form a helix bundle, which dictates lid assembly (4, 5).In the base, the six Rpt subunits form a hexameric ring. Powered by ATP hydrolysis, the Rpt ring is responsible for substrate unfolding and translocation of the unfolded substrate through the narrow RP central channel into the CP for degradation (6-8). The barrel-shaped CP consists of two outer α-rings and two inner β-rings, each containing seven subunits (α1-7 or β1-7). X-ray structures of the CP at atomic resolution have been reported for archaeabacteria (9), yeast (10), and mammals (11).Crystallization of the RP or the 26S proteasome is hampered by its dynamic nature. Improvement of cryo-EM technologies has ...

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Structural characterization of the interaction of Ubp6 with the 26S proteasome

Cited by 100 publications

References 41 publications

Structural insights into the functional cycle of the ATPase module of the 26S proteasome

Structural insights into the functional cycle of the ATPase module of the 26S proteasome

Characterization of Dynamic UbR-Proteasome Subcomplexes by In vivo Cross-linking (X) Assisted Bimolecular Tandem Affinity Purification (XBAP) and Label-free Quantitation

Structure of an endogenous yeast 26S proteasome reveals two major conformational states

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