The molecular machinery of protein degradation - structural studies ex situ and in situ

The 26S proteasome operates at the executive end of the ubiquitin-proteasome pathway for the controlled degradation of intracellular proteins. The 2.5 MDa complex is built of 34 different subunits and comprises two subcomplexes: the 20S core where proteolysis takes place and one or two regulating particles which prepare substrates for degradation. Whereas the structure of its 20S core particle has been determined by X-ray crystallography two decades ago, the structure of the 19S regulatory particle, which recruits substrates, unfolds them, and translocates them to the core particle for degradation, has been determined only in recent years.
 
Cryo electron tomography allows to perform structural studies of macromolecular and supramolecular structures in situ, i.e. in their functional cellular environments. We used this method to study 26S proteasomes in a number of cellular settings revealing their precise location, assembly and activity status as well as their interactions with other parts of the degradation machinery.