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Subunit localization of the Drosophila 26S proteasome by means of 3D cryo electron microscopy

Objective

In eukaryotic cells, the majority of cytosolic and nuclear proteins are degraded via the ubiquitin–proteasome pathway. Proteins carrying multiubiquitin tags are selected and degraded by the 26S proteasome, which is a large molecular assembly built from 30 different subunits.Two major components form the 26S complex: the proteolytic core particle (CP) and the regulatory particles (RPs).Whereas the structure and enzymatic mechanism of the CP have been studied in great detail, current understanding of the structure and function of the RP is lagging behind, together with the correct structural analysis of the 26S proteasomes. As a consequence of the low structural stability of the complex, electron micrographs of 26S preparations display structural heterogeneity that complicates image analysis and three-dimensional reconstruction. In the research proposal presented here, extensive effort will be devoted for the elucidation of the subunit topology of the RP. For this purpose, subunits will be labelled with various techniques, and their position within the 26S proteasome will be mapped by means of single particle cryo-electron microscopy. By applying 3D cryo-electron microscopy, the localisation of the labelled subunits can be reliably interpreted in terms of topology.The research project also aims to identify the subunits involved in the formation of the binding surfaces between the base and lid subcomplexes of the RP and the identification of the subunits involved in linking them. In the third stage of the project we will focus on mapping the tetraubiquitin binding site of the 26S proteasome by comparing the structure of the tetraubiquitin-26S proteasome complexes with the structure of 26S proteasome without tetraubiquitin. These approaches will allow for the first time the exact mapping of individual subunits within the RP and will allow us to better understand the interactions occurring between different subunits and between multiubiquitylated substrates and subunits.

Field of science

  • /natural sciences/mathematics/pure mathematics/topology
  • /natural sciences/biological sciences/biochemistry/biomolecules/proteins
  • /natural sciences/physical sciences/theoretical physics/particles

Call for proposal

FP7-PEOPLE-2007-2-1-IEF
See other projects for this call

Funding Scheme

MC-IEF - Intra-European Fellowships (IEF)

Coordinator

MAX-PLANCK-GESELLSCHAFT ZUR FORDERUNG DER WISSENSCHAFTEN EV
Address
Hofgartenstrasse 8
80539 Munich
Germany
Activity type
Other
EU contribution
€ 158 127,84
Administrative Contact
Ulrike Jendis (Ms.)