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

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

During the course of my Marie Curie fellowship I expertised in the laboratory headed by Prof. Wolfgang Baumeister, a leading group in the field of three-dimensional electron microscopy and in particular single particle cryo-electron microscopy (cryo-EM). Single particle cryo-EM emerged as a powerful tool for the study of macromolecular complexes preserved in their native conformation. The main goal of this fellowship was to acquire the necessary methodologies required to address fundamental biological questions using cryo-EM as a main tool. The study presented here described the localisation of the ubiquitin receptor subunit of the 26S proteasome, using single particle cryo-EM as the main research tool. The ubiquitin-proteasome system catalyses the majority of protein degradation in the eukaryotic cell. The selected proteins are marked for cellular degradation by the covalent attachment of a polyubiquitin chain. Polyubiquitinated proteins are selectively recognised and degraded by the 26S proteasome. The 26S proteasome is a large molecular assembly built from 35 different subunits that has a combined molecular mass of about 2 500 kDa. Two major components form the 26S complex: the barrel-shaped proteolytic core particle (the 20S proteasome or CP) and the regulatory particle (the 19S complex or RP), which associates with either one or both ends of the core particle. The RPs ensure the selectivity of the degradation by recognising proteins carrying polyubiquitin tags, catalyse deubiquitination, unfolding of the substrates, and finally, translocation of the unfolded substrates into the 20S complex, where the proteins are degraded into small peptides. Progress in determining the structure of the 26S proteasome has been hampered by the low intrinsic stability of the holocomplex, which tends to dissociate during purification and EM sample preparation. Application of single-particle cryo-EM and automated data acquisition procedure revealed a huge step forward in the structure determination of the proteasome. Even more detailed structural insights were obtained from cryo-EM reconstructions at a resolution of approximately 20 Angstrom. Despite of these improvements the resolution is still not good enough to outline and assign subunits of the RP to its 3D structure. Therefore the aim of my project was to localise different RP subunits within the 26S complex by subunit specific labelling. For this purpose, after purification of the holocomplex, the subunits were planned to be labelled with subunit specific antibodies, interacting proteins or ligands, and their coordinates within the 26S proteasome were planned to be mapped by means of single-particle cryo-EM. For immune labelling, we have tried several mono- and polyclonal antibodies raised against different RP subunits of the Drosophila melanogaster proteasome. The antibodies have been proven to be specific for the given subunits; however we couldn't detect by cryo-EM on the 26S proteasome binding of any of the RP subunit specific antibodies tested. Either the epitop of them was not accessible in the context of the 26S complex or the incubation with them caused the disassembly of the 26S even at 4C. In a previous study of our lab, advanced image classification revealed an extra density of 60 +/- 25 kDa which has been found in approximately 25 % of the RPs of the analysed 26S complexes. Quantitative mass spectrometry of conventionally purified Drosophila melanogaster proteasomes suggested that the ubiquitin receptor subunit of the proteasome (Rpn10) is present also in only 25 % of the RPs, which indicated that the extra density may correspond to Rpn10. To corroborate this hypothesis we have developed and affinity purification method, which allowed us to specifically label the Rpn10 subunit of the proteasome. This method is based on the specific interaction of a proteasome interacting protein, Dsk2, via the Rpn10 subunit, with the 26S proteasome.

Текст от CORDIS, на английски · Данни: CORDIS, © Европейски съюз