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 Hrr25 triggers selective autophagy–related pathways by phosphorylating receptor proteins

2014 ◽  
Vol 207 (1) ◽  
pp. 91-105 ◽  
Author(s):  
Chikara Tanaka ◽  
Li-Jing Tan ◽  
Keisuke Mochida ◽  
Hiromi Kirisako ◽  
Michiko Koizumi ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Molecular Basis ◽  
Receptor Proteins ◽  
Selective Autophagy ◽  
The Core ◽  
The Common ◽  
Related Proteins

In selective autophagy, degradation targets are specifically recognized, sequestered by the autophagosome, and transported into the lysosome or vacuole. Previous studies delineated the molecular basis by which the autophagy machinery recognizes those targets, but the regulation of this process is still poorly understood. In this paper, we find that the highly conserved multifunctional kinase Hrr25 regulates two distinct selective autophagy–related pathways in Saccharomyces cerevisiae. Hrr25 is responsible for the phosphorylation of two receptor proteins: Atg19, which recognizes the assembly of vacuolar enzymes in the cytoplasm-to-vacuole targeting pathway, and Atg36, which recognizes superfluous peroxisomes in pexophagy. Hrr25-mediated phosphorylation enhances the interactions of these receptors with the common adaptor Atg11, which recruits the core autophagy-related proteins that mediate the formation of the autophagosomal membrane. Thus, this study introduces regulation of selective autophagy as a new role of Hrr25 and, together with other recent studies, reveals that different selective autophagy–related pathways are regulated by a uniform mechanism: phosphoregulation of the receptor–adaptor interaction.

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.

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 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 Nucleotide-Driven Triple-State Remodeling of the AAA-ATPase Channel in the Activated Human 26S Proteasome

2017 ◽  
Author(s):  
Yanan Zhu ◽  
Wei Li Wang ◽  
Daqi Yu ◽  
Qi Ouyang ◽  
Ying Lu ◽  
...  
Keyword(s):  
Conformational Changes ◽  
26S Proteasome ◽  
Eukaryotic Cells ◽  
Alternative States ◽  
Nucleotide Exchange ◽  
Core Particle ◽  
Aaa Atpase ◽  
The Core ◽  
Gate Opening ◽  
Proteolytic Chamber

SUMMARYThe proteasome is a sophisticated ATP-dependent molecular machine responsible for protein degradation in all eukaryotic cells. It remains elusive how conformational changes of the AAA-ATPase unfoldase in the regulatory particle (RP) control the gating of substrate-translocation channel to the proteolytic chamber of the core particle (CP). Here we report three alternative states of the ATP-γS-bound human proteasome, in which the CP gate is asymmetrically open, visualized by cryo-EM at near-atomic resolutions. Only four nucleotides are stably bound to the AAA-ATPase ring in the open-gate states. Concerted nucleotide exchange gives rise to a back-and-forth wobbling motion of the AAA-ATPase channel, coincident with remarkable transitions of their pore loops between the spiral staircase and saddle-shaped circle topologies. Gate opening in the CP is thus controlled with nucleotide-driven remodeling of the AAA-ATPase unfoldase. These findings demonstrate an elegant mechanism of allosteric coordination among sub-machines within the holoenzyme that is crucial for substrate translocation.

scholarly journals Yeast Rpb9 Plays an Important Role in Ubiquitylation and Degradation of Rpb1 in Response to UV-Induced DNA Damage

2007 ◽  
Vol 27 (13) ◽  
pp. 4617-4625 ◽  
Author(s):  
Xuefeng Chen ◽  
Christine Ruggiero ◽  
Shisheng Li
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Damage ◽  
Rna Polymerase Ii ◽  
Uv Radiation ◽  
Excision Repair ◽  
Transcription Elongation ◽  
26S Proteasome ◽  
Pol Ii ◽  
The Core ◽  
Nucleotide Excision

ABSTRACT Rpb9, a nonessential subunit of RNA polymerase II (Pol II), has multiple transcription-related functions in Saccharomyces cerevisiae, including transcription elongation and transcription-coupled repair (TCR). Here we show that, in response to UV radiation, Rpb9 also functions in promoting ubiquitylation and degradation of Rpb1, the largest subunit of Pol II. This function of Rpb9 is not affected by any pathways of nucleotide excision repair, including TCR mediated by Rpb9 itself and by Rad26. Rpb9 is composed of three distinct domains: the N-terminal Zn1, the C-terminal Zn2, and the central linker. The Zn2 domain, which is dispensable for transcription elongation and TCR functions, is essential for Rpb9 to promote Rpb1 degradation, whereas the Zn1 and linker domains, which are essential for transcription elongation and TCR functions, play a subsidiary role in Rpb1 degradation. Coimmunoprecipitation analysis suggests that almost the full length of Rpb9 is required for a strong interaction with the core Pol II: deletion of the Zn2 domain causes dramatically weakened interaction, whereas deletion of Zn1 and the linker resulted in undetectable interaction. Furthermore, we show that Rpb1, rather than the whole Pol II complex, is degraded in response to UV radiation and that the degradation is primarily mediated by the 26S proteasome.

scholarly journals Molecular Basis of Fructose Utilization by the Wine Yeast Saccharomyces cerevisiae: a Mutated HXT3 Allele Enhances Fructose Fermentation

2007 ◽  
Vol 73 (8) ◽  
pp. 2432-2439 ◽  
Author(s):  
Carole Guillaume ◽  
Pierre Delobel ◽  
Jean-Marie Sablayrolles ◽  
Bruno Blondin
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Molecular Basis ◽  
Alcoholic Fermentation ◽  
Wine Yeast ◽  
Glycolytic Flux ◽  
Hexose Transporter ◽  
Wine Yeasts ◽  
Yeast Saccharomyces Cerevisiae ◽  
High Fructose ◽  
Fructose Fermentation

ABSTRACT Fructose utilization by wine yeasts is critically important for the maintenance of a high fermentation rate at the end of alcoholic fermentation. A Saccharomyces cerevisiae wine yeast able to ferment grape must sugars to dryness was found to have a high fructose utilization capacity. We investigated the molecular basis of this enhanced fructose utilization capacity by studying the properties of several hexose transporter (HXT) genes. We found that this wine yeast harbored a mutated HXT3 allele. A functional analysis of this mutated allele was performed by examining expression in an hxt1-7Δ strain. Expression of the mutated allele alone was found to be sufficient for producing an increase in fructose utilization during fermentation similar to that observed in the commercial wine yeast. This work provides the first demonstration that the pattern of fructose utilization during wine fermentation can be altered by expression of a mutated hexose transporter in a wine yeast. We also found that the glycolytic flux could be increased by overexpression of the mutant transporter gene, with no effect on fructose utilization. Our data demonstrate that the Hxt3 hexose transporter plays a key role in determining the glucose/fructose utilization ratio during fermentation.

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