Dr. Eri Sakata
Phone:+49 89 8578 2648

Sakata-CV

Members

Florian Beck
Email:fbeck@...
Günter Pfeifer
Dr. Parijat Majumder
Dr. Andreas Schweitzer
Dr. Marc Wehmer
Markus Eisele

Alumni

Dr. Nicole Eisele
Oana Mihalache
Dr. Ganesh Pathare
Dr. Paweł Śledź
Dr. Stefan Bohn
Dr. Stephan Nickell

Proteasome

Structural dynamics at the 26S Proteasome

<p>(A) Electron micrograph of the 26S proteasome embedded in vitreous ice. (B) 4.1 Å resolution EM single particle reconstruction of S.cerevisiae 26S proteasome. Wehmer, et al., PNAS (2017)</p> Zoom Image

(A) Electron micrograph of the 26S proteasome embedded in vitreous ice. (B) 4.1 Å resolution EM single particle reconstruction of S.cerevisiae 26S proteasome. Wehmer, et al., PNAS (2017)

[less]

The ubiquitin proteasome system (UPS) is a central pathway of protein degradation in the cell, regulating protein homeostasis and quality-control. The 26S proteasome is 2.5 MDa giant protease responsible for the regulated degradation of polyubiquitylated proteins. It consists of at least 33 distinct subunits and is arranged into two modules; core particle (CP) containing catalytic sites and regulatory particles (RP). The cylinder-shaped proteolytic core is the 20S CP, which is capped at one or both ends by 19S RPs.

The structure of the 26S holocomplex has remained elusive for decades despite its essential role in the maintenance of protein homeostasis. We have revealed the molecular architecture of the 26S proteasome with subnanometer resolution using cryo-EM single particle analysis (Figure 1). We elucidated the localization of the individual subunits of the 19S RP and provided a pseudo-atomic structure of the 26S proteasome. A recent deep classification approach of a large cryo-EM dataset enabled us to deconvolute hidden conformational states. The resulting conformational ensemble of the 26S proteasome provides insights into the mechanisms of substrate processing in coupled with the gate-opening mechanism of the 20S CP (Figure 2 and movie).

To further understand how the 26S proteasome executes its function, we take a multidisciplinary approach combining cryo-EM single particle analysis with biochemical and computational studies. We aim to elucidate the structural basis of substrate processing and the regulatory mechanisms by its cofactors. At the same time, cryo-EM technology has been revolutionized by automated data acquisition schemes and new electron detectors, which empower us to explore dynamic structures at high resolution. Thus, we aim not only “watch” conformational changes in macromolecules as they perform their function, but also structurally analyze their transient interactions.

Recently, our group developed a method to visualize the 26S proteasome in situ by cryo-electron tomography (cryo-ET) combining a new type of phase plate and direct detectors.  This technique allows us to study the molecular architecture of the 26S proteasome in its cellular environment. This study will help us to understand the structural dynamics of the 26S proteasome in situ.

<p>Functional model for substrate degradation.  Unverdorben, Beck et al , PNAS (2014)</p> Zoom Image

Functional model for substrate degradation.  Unverdorben, Beck et al , PNAS (2014)

 

Fundings

https://www.sfb1035.tum.de/en/start/

Publications

Wehmer M, Rudack T, Beck F, Aufderheide A, Pfeifer G, Plitzko JM, Förster F, Schulten K, Baumeister W, Sakata E. Structural insights into the functional cycle of the ATPase module of the 26S proteasome.  Proc Natl Acad Sci U S A. 114(6):1305-1310, 2017

Wehmer M and Sakata E. Recent advances in the structural biology of the 26S proteasome. Int J Biochem Cell Biol. 79:437-442, 2016

Schweitzer A, Aufderheide A, Rudack T, Beck F, Pfeifer G, Plitzko JM, Sakata E, Schulten K, Förster F, Baumeister W. Structure of the human 26S proteasome at a resolution of 3.9 Å. Proc Natl Acad Sci U S A. 113(28):7816-21, 2016

Aufderheide A, Beck F, Stengel F, Hartwig M, Schweitzer A, Pfeifer G, Goldberg AL, Sakata E, Baumeister W, Förster F. Structural characterization of the interaction of Ubp6 with the 26S proteasome. Proc Natl Acad Sci U S A. 112 (28):8626-31, 2015

Asano S, Fukuda Y, Beck F, Aufderheide A, Förster F, Danev R, Baumeister W. Proteasomes. A molecular census of 26S proteasomes in intact neurons. Science. 347(6220):439-42, 2015

Unverdorben P, Beck F, Śledź P, Schweitzer A, Pfeifer G, Plitzko JM, Baumeister W, Förster F. Deep classification of a large cryo-EM dataset defines the conformational landscape of the 26S proteasome. Proc. Natl. Acad. Sci. USA. 111:5544-5549, 2014

 

Pathare GR, Nagy I, Śledź P, Anderson DJ, Zhou HJ, Pardon E, Steyaert J, Förster F, Bracher A, Baumeister W. Crystal structure of the proteasomal deubiquitylation module Rpn8-Rpn11. Proc. Natl. Acad. Sci. USA. 111:2984-2989, 2014

 

Sledz P., Unverdorben P., Beck F., Pfeifer G., Schweitzer A., Förster F. and Baumeister W.: Structure of the 26S proteasome with ATP-γS bound provides insights into the mechanism of nucleotide-dependent substrate translocation. Proc. Natl. Acad. Sci. USA. 110:7264-7269, 2013

 

Beck F., Unverdorben P., Bohn S., Schweitzer A., Pfeifer G., Sakata E., Nickell S., Plitzko J.M., Villa E., Baumeister W. and Förster F.: Near-atomic resolution structural model of the yeast 26S proteasome. Proc. Natl. Acad. Sci. USA. 109:14870-14875, 2012

 

Lasker K., Förster F., Bohn S., Walzthoeni T., Villa E., Unverdorben P., Beck F., Aebersold R., Sali A. and Baumeister W.: Molecular architecture of the 26S proteasome holocomplex determined by an integrative approach. Proc. Natl. Acad. Sci. USA. 109:1380-7, 2012

 

Sakata E., Bohn S., Mihalache O., Kiss P., Beck F., Nagy I., Nickell S., Tanaka K., Saeki Y., Förster F. and Baumeister W.: Localization of the proteasomal ubiquitin receptors Rpn10 and Rpn13 by electron cryomicroscopy. Proc. Natl. Acad. Sci. USA. 109:1479-84, 2012

 

Pathare G.R., Nagy I., Bohn S., Unverdorben P., Hubert A., Körner R., Nickell S., Lasker K., Sali A., Tamura T., Nishioka T., Förster F. and Baumeister W.: Bracher A, The proteasomal subunit Rpn6 is a molecular clamp holding the core and regulatory subcomplexes together. Proc. Natl. Acad. Sci. USA. 109(1):149-54, 2012

 

Sakata E, Stengel F, Fukunaga K, Zhou M, Saeki Y, Förster F, Baumeister W, Tanaka K, Robinson CV. The catalytic activity of Ubp6 enhances maturation of the proteasomal regulatory particle. Mol Cell 42:637-649, 2011

 

Bohn S., Beck F., Sakata E., Walzthoeni T., Beck M., Aebersold R., Förster F., Baumeister W. and Nickell S.: Structure of the 26S proteasome from Schizosaccharomyces pombe at sub-nanometer resolution. Proc. Natl. Acad. Sci. USA 107:20992-7, 2010

 

Förster F, Lasker K, Beck F, Nickell S, Sali A, Baumeister W. An atomic model AAA-ATPase/20S core particle sub-complex of the 26S proteasome. BBRC 388(2):228-33, 2009

 

Nickell S., Beck F., Scheres S.H., Korinek A., Förster F., Lasker K., Mihalache O., Sun N., Nagy I., Sali A., Plitzko J.M., Carazo J.M., Mann M. and Baumeister W.: Insights into the molecular architecture of the 26S proteasome. Proc Natl Acad Sci U S A. 106(29):11943-7, 2009

 

Hölzl H, Kapelari B, Kellermann J, Seemüller E, Sümegi M, Udvardy A, Medalia O, Sperling J, Müller SA, Engel A, Baumeister W. The regulatory complex of Drosophila melanogaster 26S proteasomes. Subunit composition and localization of a deubiquitylating enzyme. J Cell Biol. 150(1):119-30, 2000

 

Walz J, Erdmann A, Kania M, Typke D, Koster AJ, Baumeister W. 26S proteasome structure revealed by three-dimensional electron microscopy. J Struct Biol. 121(1):19-29, 1998

 

Zwickl P, Pfeifer G, Lottspeich F, Kopp F, Dahlmann B, Baumeister W. Electron microscopy and image analysis reveal common principles of organization in two large protein complexes: groEL-type proteins and proteasomes. J Struct Biol. 103(3):197-203, 1990

 

Dahlmann B, Kopp F, Kuehn L, Niedel B, Pfeifer G, Hegerl R, Baumeister W. The multicatalytic proteinase (prosome) is ubiquitous from eukaryotes to archaebacteria. FEBS Lett. 251(1-2):125-31, 1989

 

Baumeister W, Dahlmann B, Hegerl R, Kopp F, Kuehn L, Pfeifer G. Electron microscopy and image analysis of the multicatalytic proteinase. FEBS Lett. 241(1-2):239-45, 1988

 
loading content