26S PROTEASOME

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

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 Obiettivo del progetto (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.'

Introduzione (Teaser)

Large protein complexes such as the 26S proteasome have been widely studied. Their two major components are the core particle and the regulatory particle.

Descrizione progetto (Article)

The 26S proteasome is a large mass of molecules made up of some 35 subunits. While the proteolytic core particle (CP) has been extensively researched, there is a lack of knowledge about the regulatory particle (RP).

The EU-funded project 'Subunit localisation of the Drosophila 26S proteasome by means of 3D cryo electron microscopy' (26S Proteasome) set out to study the makeup of the RP's subunits. To do this, researchers used various techniques to label subunits, and map their position within the 26S proteasome by single particle cryo-electron microscopy (CEM). This is a powerful way of studying the complex's three-dimensional (3D) organisation. However, the resolution is still inadequate to fully outline and assign RP subunits.

During the first phase of the project, three chromatographic steps were used to purify the 26S proteasome ahead of antibody labelling. One CP subunit and five RP subunits were successfully labelled. In the second phase, the 26S Proteasome project focused on localising the Rpn10 subunit, for which a new purification and labelling method had to be developed. The particular method provides means for one-step affinity purification, and the proteasomes are labelled with the help of the specific interaction partner.

Project members used biochemical techniques to analyse the structural integrity, proteolytic activity and subunit reactions of the affinity-isolated and labelled proteasomes, which were then used to perform single particle CEM analysis. This gave researchers the means of mapping the attachment site of the nuclear-enriched protein Dsk2 on the 26S proteasome complexes and ultimately localising the Rpn10 subunit.