ABSTRACT
T he cytoplasm of mammalian tissues contains high concentrations of 20S proteasomes, the core of the major cytosolic proteolytic system.1 This multicatalytic proteinase degrades proteins into oligopeptides of about 3-15 amino acids.2,3 To protect cellular proteins from uncontrolled shredding by the enzyme, nature has developed effective mechanisms. The main protection mechanism lies in the structure of proteasomes themselves. They are cylinder-shaped particles consisting of 24 proteins arranged in four stacked seven-membered rings.4'6 The closing off rings are built up by a subunits whereas each of the two adjacent central rings is composed of β subunits. Cavities are formed between the a - and β -rings and in the proteasome centre between the two β rings. As the hydroxyl groups of the N-terminal threonine residues of several β subunits, which have been found to function as active site nucleophiles, are exposed to the central cavity,7 cellular proteins do not risk to be degraded as long as they have not entered this proteolytic compartment. Rather, substrate proteins have to be unfolded before they can enter the barrel like proteasome complexes and get access to their active sites.8 During biogenesis of 20S proteasomes uncontrolled proteolysis is avoided by synthesis of inactive precursor subunits that are proteolytically activated only as the active sites become caged in the
assembled proteasome (for a review see reference 9). The architecture of proteasomes has been conserved during evolution of all three kingdoms of living organisms. While archae-and eubacterial proteasomes contain only one or two different a and β type subunits (for a review see reference 9), yeast proteasomes are composed of seven different a type and seven different β type subunits.10 Even 10 different kinds o f β subunits have been detected in proteasomes of mammalian cells.11 Since there is evidence that all subunits occupy defined positions in a proteasome cylinder, we have investigated their arrangement in the human 20S proteasome by localizing them with electron microscopic methods as already used successfully for Thermoplasma protea somes12 and by determination of neighboring subunits by chemical crosslinking. Knowledge of the subunit arrangement is necessary in order to understand the function of the 20S proteasom e itse lf and of the regulator complexes that associate with the proteasome. Such regulators induce enhancement and modulation of its activity or enable binding and u n fo ld in g o f p ro teins w hich are committed to degradation by posttranslational polyubiquitination (for review see references 13-15). Additionally, protein inhibitors exist that may attenuate the activities of the proteasomal system in the cell.13
