scholarly journals The Assembly Pathway of the 19S Regulatory Particle of the Yeast 26S Proteasome

2007 ◽  
Vol 18 (2) ◽  
pp. 569-580 ◽  
Author(s):  
Erika Isono ◽  
Kiyoshi Nishihara ◽  
Yasushi Saeki ◽  
Hideki Yashiroda ◽  
Naoko Kamata ◽  
...  
Keyword(s):  
26S Proteasome ◽  
Restrictive Temperature ◽  
Enzyme Complex ◽  
Core Particle ◽  
Yeast Saccharomyces Cerevisiae ◽  
The Core ◽  
Assembly Pathway ◽  
Regulatory Particle ◽  
Mutant Cells

The 26S proteasome consists of the 20S proteasome (core particle) and the 19S regulatory particle made of the base and lid substructures, and it is mainly localized in the nucleus in yeast. To examine how and where this huge enzyme complex is assembled, we performed biochemical and microscopic characterization of proteasomes produced in two lid mutants, rpn5-1 and rpn7-3, and a base mutant ΔN rpn2, of the yeast Saccharomyces cerevisiae. We found that, although lid formation was abolished in rpn5-1 mutant cells at the restrictive temperature, an apparently intact base was produced and localized in the nucleus. In contrast, in ΔN rpn2 cells, a free lid was formed and localized in the nucleus even at the restrictive temperature. These results indicate that the modules of the 26S proteasome, namely, the core particle, base, and lid, can be formed and imported into the nucleus independently of each other. Based on these observations, we propose a model for the assembly process of the yeast 26S proteasome.

scholarly journals In-depth Analysis of the Lid Subunits Assembly Mechanism in Mammals

Biomolecules ◽  
2019 ◽  
Vol 9 (6) ◽  
pp. 213 ◽  
Author(s):  
Minghui Bai ◽  
Xian Zhao ◽  
Kazutaka Sahara ◽  
Yuki Ohte ◽  
Yuko Hirano ◽  
...  
Keyword(s):  
26S Proteasome ◽  
Core Particle ◽  
Proteasome Subunit ◽  
Ubiquitinated Proteins ◽  
Assembly Pathway ◽  
Assembly Mechanism ◽  
Depth Analysis ◽  
Regulatory Particle ◽  
Assembly Intermediate ◽  
Assembly Pathways

The 26S proteasome is a key player in the degradation of ubiquitinated proteins, comprising a 20S core particle (CP) and a 19S regulatory particle (RP). The RP is further divided into base and lid subcomplexes, which are assembled independently from each other. We have previously demonstrated the assembly pathway of the CP and the base by observing assembly intermediates resulting from knockdowns of each proteasome subunit and the assembly chaperones. In this study, we examine the assembly pathway of the mammalian lid, which remains to be elucidated. We show that the lid assembly pathway is conserved between humans and yeast. The final step is the incorporation of Rpn12 into the assembly intermediate consisting of two modular complexes, Rpn3-7-15 and Rpn5-6-8-9-11, in both humans and yeast. Furthermore, we dissect the assembly pathways of the two modular complexes by the knockdown of each lid subunit.

CDC68, a yeast gene that affects regulation of cell proliferation and transcription, encodes a protein with a highly acidic carboxyl terminus

1991 ◽  
Vol 11 (11) ◽  
pp. 5718-5726
Author(s):  
A Rowley ◽  
R A Singer ◽  
G C Johnston
Keyword(s):  
Gene Expression ◽  
Cell Cycle ◽  
Cycle Performance ◽  
Carboxyl Terminus ◽  
Temperature Sensitive ◽  
Restrictive Temperature ◽  
Negative Effects ◽  
Yeast Saccharomyces Cerevisiae ◽  
Acid Protein ◽  
Mutant Cells

The cell cycle of the budding yeast Saccharomyces cerevisiae has been investigated through the study of conditional cdc mutations that specifically affect cell cycle performance. Cells bearing the cdc68-1 mutation (J. A. Prendergast, L. E. Murray, A. Rowley, D. R. Carruthers, R. A. Singer, and G. C. Johnston, Genetics 124:81-90, 1990) are temperature sensitive for the performance of the G1 regulatory event, START. Here we describe the CDC68 gene and present evidence that the CDC68 gene product functions in transcription. CDC68 encodes a 1,035-amino-acid protein with a highly acidic and serine-rich carboxyl terminus. The abundance of transcripts from several unrelated genes is decreased in cdc68-1 mutant cells after transfer to the restrictive temperature, while at least one transcript, from the HSP82 gene, persists in an aberrant fashion. Thus, the cdc68-1 mutation has both positive and negative effects on gene expression. Our findings complement those of Malone et al. (E. A. Malone, C. D. Clark, A. Chiang, and F. Winston, Mol. Cell. Biol. 11:5710-5717, 1991), who have independently identified the CDC68 gene (as SPT16) as a transcriptional suppressor of delta-insertion mutations. Among transcripts that rapidly become depleted in cdc68-1 mutant cells are those of the G1 cyclin genes CLN1, CLN2, and CLN3/WHI1/DAF1, whose activity has been previously shown to be required for the performance of START. The decreased abundance of cyclin transcripts in cdc68-1 mutant cells, coupled with the suppression of cdc68-1-mediated START arrest by the CLN2-1 hyperactive allele of CLN2, shows that the CDC68 gene affects START through cyclin gene expression.

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

2017 ◽  
Vol 114 (6) ◽  
pp. 1305-1310 ◽  
Author(s):  
Marc Wehmer ◽  
Till Rudack ◽  
Florian Beck ◽  
Antje Aufderheide ◽  
Günter Pfeifer ◽  
...  
Keyword(s):  
Atp Hydrolysis ◽  
Ubiquitin Proteasome System ◽  
26S Proteasome ◽  
Core Particle ◽  
Nucleotide Analogs ◽  
The Core ◽  
Cryo Electron Microscopy ◽  
Intracellular Proteins ◽  
Ubiquitin Proteasome ◽  
Structural Insights

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.

Chaperone-driven proteasome assembly

2008 ◽  
Vol 36 (5) ◽  
pp. 807-812 ◽  
Author(s):  
Rina Rosenzweig ◽  
Michael H. Glickman
Keyword(s):  
26S Proteasome ◽  
Biologically Active ◽  
Core Particle ◽  
Regulatory Particle ◽  
Particle Attachment

Assembly of the 34-subunit, 2.5 MDa 26S proteasome is a carefully choreographed intricate process. It starts with formation of a seven-membered α-ring that serves as a template for assembly of the complementary β-ring-forming ‘half-proteasomes’. Dimerization results in a latent 20S core particle that can serve further as a platform for 19S regulatory particle attachment and formation of the biologically active 26S proteasome for ubiquitin-dependent proteolysis. Both general and dedicated proteasome assembly chaperones regulate the efficiency and outcome of critical steps in proteasome biogenesis, and in complex association.

scholarly journals Assembly manual for the proteasome regulatory particle: the first draft

2010 ◽  
Vol 38 (1) ◽  
pp. 6-13 ◽  
Author(s):  
Soyeon Park ◽  
Geng Tian ◽  
Jeroen Roelofs ◽  
Daniel Finley
Keyword(s):  
Biochemical Study ◽  
Release Mechanism ◽  
Substrate Selection ◽  
Core Particle ◽  
Genetic Studies ◽  
Macromolecular Assembly ◽  
Assembly Pathway ◽  
Ongoing Work ◽  
The Common ◽  
Regulatory Particle

The proteasome is the most complex protease known, with a molecular mass of approx. 3 MDa and 33 distinct subunits. Recent studies reported the discovery of four chaperones that promote the assembly of a 19-subunit subcomplex of the proteasome known as the regulatory particle, or RP. These and other findings define a new and highly unusual macromolecular assembly pathway. The RP mediates substrate selection by the proteasome and injects substrates into the CP (core particle) to be degraded. A heterohexameric ring of ATPases, the Rpt proteins, is critical for RP function. These ATPases abut the CP and their C-terminal tails help to stabilize the RP–CP interface. ATPase heterodimers bound to the chaperone proteins are early intermediates in assembly of the ATPase ring. The four chaperones have the common feature of binding the C-domains of Rpt proteins, apparently a remarkable example of convergent evolution; each chaperone binds a specific Rpt subunit. The C-domains are distinct from the C-terminal tails, but are proximal to them. Some, but probably not all, of the RP chaperones appear to compete with CP for binding of the Rpt proteins, as a result of the proximity of the tails to the C-domain. This competition may underlie the release mechanism for these chaperones. Genetic studies in yeast point to the importance of the interaction between the CP and the Rpt tails in assembly, and a recent biochemical study in mammals suggests that RP assembly takes place on pre-assembled CP. These results do not exclude a parallel CP-independent pathway of assembly. Ongoing work should soon clarify the roles of both the CP and the four chaperones in RP assembly.

Chaperone-assisted assembly of the proteasome core particle

2010 ◽  
Vol 38 (1) ◽  
pp. 29-33 ◽  
Author(s):  
Ana C. Matias ◽  
Paula C. Ramos ◽  
R. Jürgen Dohmen
Keyword(s):  
Active Sites ◽  
26S Proteasome ◽  
Eukaryotic Cells ◽  
Main Function ◽  
Core Particle ◽  
Lysosomal Protease ◽  
Assembly Pathway ◽  
Multimeric Complex ◽  
Autocatalytic Processing ◽  
Α Subunits

The 26S proteasome is a non-lysosomal protease in the cytosol and nucleus of eukaryotic cells. Its main function is to mediate ubiquitin-dependent proteolysis. The 26S proteasome is a multimeric complex composed by the 20S proteasome CP (core particle) and the 19S RPs (regulatory particles). Although the atomic structure of the 26S proteasome has not yet been determined, high-resolution structures are available for its CP. Studies on the complicated assembly pathway of the proteasome have revealed that it involves an unprecedented number of dedicated chaperones. Assembly of the CP alone involves three conserved proteasome-assembly chaperones [PAC1–PAC2, PAC3–PAC4 and UMP1 (ubiquitin-mediated proteolysis 1)]. Whereas the two heterodimeric PACs have been implicated in the formation of rings of the seven distinct α subunits, UMP1 is important for the formation and dimerization of proteasome precursor complexes containing β subunits. Dimerization coincides with the incorporation of the last β subunit (β7). Additional modules important for the assembly of precursor complexes and their dimerization reside in the β subunits themselves, either as transient or as permanent extensions. Particularly important domains are the propeptide of β5 and the C-terminal extensions of β2 and β7. Upon maturation of the active sites by autocatalytic processing, UMP1 is degraded by the native proteasome.

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

2016 ◽  
Vol 113 (10) ◽  
pp. 2642-2647 ◽  
Author(s):  
Bai Luan ◽  
Xiuliang Huang ◽  
Jianping Wu ◽  
Ziqing Mei ◽  
Yiwei Wang ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Enzyme Activity ◽  
Molecular Basis ◽  
Biochemical Analysis ◽  
26S Proteasome ◽  
Core Particle ◽  
Conformational States ◽  
The Core ◽  
Substrate Structure ◽  
Exogenous Substrate

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.

scholarly journals Preliminary characterization of maturation-promoting factor from yeast Saccharomyces cerevisiae

1987 ◽  
Vol 88 (3) ◽  
pp. 273-281
Author(s):  
K. Tachibana ◽  
N. Yanagishima ◽  
T. Kishimoto
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Small Molecules ◽  
Sedimentation Coefficient ◽  
Restrictive Temperature ◽  
Dependent Manner ◽  
Yeast Saccharomyces Cerevisiae ◽  
Preliminary Characterization ◽  
M Phase ◽  
Maturation Promoting Factor

It has been known for some time that maturation-promoting factor (MPF) appears in a wide variety of eukaryotic cells at M phase and exerts equal M-phase-promoting activity in both meiotic cells and mitotic cells in a non-specific manner. MPF was extracted from cdc20 mutant cells of the yeast Saccharomyces cerevisiae synchronized at M phase by incubation at the restrictive temperature. When injected into immature oocytes of Xenopus laevis, yeast MPF caused meiosis reinitiation in a dose-dependent manner and even in the presence of cycloheximide. Yeast MPF exerted its activity in starfish oocytes as well. MPF activity was obtained only from cells in M phase and not from G1, S or G2 phase cells, indicating cyclical changes during the yeast mitotic cell cycle. Preliminary characterization of yeast MPF revealed that its activity was associated with a heat-labile protein having a sedimentation coefficient value of 6 S. In contrast to the current assumption that MPF is a Ca-sensitive phosphoprotein stabilized by phosphorylated small molecules, such as ATP and Na-beta-glycerophosphate, the present study revealed that yeast MPF was still active even after treatment with either Ca2+ or alkaline phosphatase. Furthermore, it was found that yeast MPF and these phosphorylated small molecules were complementary in inducing reinitiation of meiosis, since the meiosis-reinitiating activity was detected only when both were present simultaneously and almost undetectable when either of them was present alone.(ABSTRACT TRUNCATED AT 250 WORDS)

scholarly journals Structural basis for dynamic regulation of the human 26S proteasome

2016 ◽  
Vol 113 (46) ◽  
pp. 12991-12996 ◽  
Author(s):  
Shuobing Chen ◽  
Jiayi Wu ◽  
Ying Lu ◽  
Yong-Bei Ma ◽  
Byung-Hoon Lee ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Ground State ◽  
Atp Hydrolysis ◽  
26S Proteasome ◽  
Structural Basis ◽  
Core Particle ◽  
Dynamic Regulation ◽  
Aaa Atpase ◽  
The Core ◽  
Aaa Atpases

The proteasome is the major engine of protein degradation in all eukaryotic cells. At the heart of this machine is a heterohexameric ring of AAA (ATPases associated with diverse cellular activities) proteins that unfolds ubiquitylated target proteins that are concurrently translocated into a proteolytic chamber and degraded into peptides. Using cryoelectron microscopy, we determined a near–atomic-resolution structure of the 2.5-MDa human proteasome in its ground state, as well as subnanometer-resolution structures of the holoenzyme in three alternative conformational states. The substrate-unfolding AAA-ATPase channel is narrowed by 10 inward-facing pore loops arranged into two helices that run in parallel with each other, one hydrophobic in character and the other highly charged. The gate of the core particle was unexpectedly found closed in the ground state and open in only one of the alternative states. Coordinated, stepwise conformational changes of the regulatory particle couple ATP hydrolysis to substrate translocation and regulate gating of the core particle, leading to processive degradation.

scholarly journals Atg17 Functions in Cooperation with Atg1 and Atg13 in Yeast Autophagy

2005 ◽  
Vol 16 (5) ◽  
pp. 2544-2553 ◽  
Author(s):  
Yukiko Kabeya ◽  
Yoshiaki Kamada ◽  
Misuzu Baba ◽  
Hirosato Takikawa ◽  
Mitsuru Sasaki ◽  
...  
Keyword(s):  
Kinase Activity ◽  
Biochemical Characterization ◽  
Eukaryotic Cells ◽  
Autophagosome Formation ◽  
Yeast Saccharomyces Cerevisiae ◽  
Starvation Condition ◽  
Mutant Cells ◽  
Two Hybrid ◽  
Starvation Conditions

In eukaryotic cells, nutrient starvation induces the bulk degradation of cellular materials; this process is called autophagy. In the yeast Saccharomyces cerevisiae, most of the ATG (autophagy) genes are involved in not only the process of degradative autophagy, but also a biosynthetic process, the cytoplasm to vacuole (Cvt) pathway. In contrast, the ATG17 gene is required specifically in autophagy. To better understand the function of Atg17, we have performed a biochemical characterization of the Atg17 protein. We found that the atg17Δ mutant under starvation condition was largely impaired in autophagosome formation and only rarely contained small autophagosomes, whose size was less than one-half of normal autophagosomes in diameter. Two-hybrid analyses and coimmunoprecipitation experiments demonstrated that Atg17 physically associates with Atg1-Atg13 complex, and this binding was enhanced under starvation conditions. Atg17-Atg1 binding was not detected in atg13Δ mutant cells, suggesting that Atg17 interacts with Atg1 through Atg13. A point mutant of Atg17, Atg17C24R, showed reduced affinity for Atg13, resulting in impaired Atg1 kinase activity and significant defects in autophagy. Taken together, these results indicate that Atg17-Atg13 complex formation plays an important role in normal autophagosome formation via binding to and activating the Atg1 kinase.

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