The 20S Proteasome
Phone: +49 - 89 - 8578 2648
Alumni:
Dr. Karin Felderer
Sarah Breuer
Dr. Bing K. Jap, LBNL Berkeley, USA Prof. Carol V. Robinson, University of Cambridge, UK Prof. Lewis E. Kay, University of Toronto, Canada
The proteasome is an ubiquitous, macromolecular assembly designed for the controlled proteolysis of either abnormal or short-lived regulatory proteins. In eukaryotes the 20S proteasome is the core complex of the 26S proteasome, which degrades ubiquitinated target molecules in an ATP-dependent manner. 20S proteasomes are found in all three urkingdoms: archaea, bacteria and eukarya. The quaternary structure is highly conserved. In electron micrographs 20S proteasomes from eukaryotes and prokaryotes are indistinguishable and appear as barrel-shaped complexes, composed of 28 subunits. The main difference between the prokaryotic and eukaryotic 20S proteasomes is one of subunit complexity. The 20S complex is composed of four stacked subunit rings. The two outer rings are formed by α-subunits, sandwiching the two inner rings, which are composed of the β-subunits. The four rings form a cylinder that is traversed by a central channel. Access to the channel is restricted by an orifice in the α-rings. The central channel widens into three chambers namely two antechambers and one central chamber. The central chamber is formed by β-subunits and contains the active sites. This self-compartimentalizing structure prevents the destruction of proteins not destined for degradation.
The mature active state of the 20S proteasome is formed through a folding and assembly pathway which, in the case of the eukaryotic proteasome, must orchestrate the correct positioning of two copies each of seven different α- and seven different β-type subunits. During the course of the assembly, β-type subunits are processed by an autocatalytic mechanism that removes the propeptide and exposes the catalytic nucleophile (i.e. the N-terminal threonine). For dissecting the assembly pathway we have used the 20S proteasome from the actinomycetes Rhodococcus erythropolis as a model system. The individual subunits can be expressed and purified separately. When mixed, they assemble to fully active proteasomes in vivo and in vitro. It is noteworthy that the Rhodococcus proteasome is in several respects more similar to the eukaryotic particle than to the archaeal one. Despite the low degree of sequence conservation among proteasomal propeptides, alignments of β-propeptides from actinomycetes and of those of the eukaryotic β2 subfamily show some conserved features. By extensive genetic engineering of the Rhodococcus subunits we were able to trap different assembly intermediates. Their characterization by biophysical methods, in particular x-ray crystallography, cryo-electron microscopy and mass spectrometry, enabled us to obtain detailed insight into some key events of the proteasome biogenesis.
The uptake of substrate by the proteasome is a multi-step process. It involves the recognition of proteins targeted for degradation, the preparation for entry by unfolding them, the gating of the orifice in the α-rings and, finally, the translocation of the substrate into the central proteolytic chamber. Regulatory complexes such as the 19S caps in the case of the eukaryotic 26S proteasome, or PAN in the case of the archaeal proteasome play critical roles in the early steps.
For studying the interactions between 20S proteasomes and substrates “en route” to the central cavity we have used quasi-stable “host-guest” complexes. Such complexes are formed by the incubation of 20S proteasomes with denatured substrates and the subsequent trapping of these “guests” by refolding within the internal cavities of the proteasome. Structural studies of “host-guest” complexes have led to the identification of a number of residues interacting with the substrates. Mutational analysis of these interactions will be complemented by the structural characterization of the “host-guest” complexes using x-ray crystallography and cryo-electron-microscopy.
From previous work with bacterial surface proteins we have experience in using the atomic force microscope (AFM) as an imaging and as a force-measuring (force spectroscopy) instrument. We routinely perform AFM studies with 20S proteasomes and other macromolecular assemblies at high resolution in an aqueous environment. To determine the forces involved in the translocation of polypeptide chains into the interior of the 20S complex we establish an experimental set-up in which 20S proteasomes are directly immobilized on mica in an upright way. The substrate is covalently bound to the tip of the AFM cantilever and offered as bait to the densely immobilised 20S proteasomes. This approach is enabling us to probe the interactions of substrates with residues lining the path from the central gate to the catalytic cavity.
Top
Print