¨r Biochemie Am Klopferspitz 18a the chaperonins and have been referred to as "reverse chaperones" or "unfoldases" (Lupas et al., 1993). Since D-82152 Martinsried Germany their action requires the hydrolysis of ATP, protein degradation becomes energy-dependent, although the hydrolysis of the polypeptide chain itself is an exergonic Controlling Proteolysis process. through CompartmentalizationSelf-compartmentalizing proteases are common in all Protein degradation is a necessity for many reasons: three domains of life: archaea, bacteria, and eukarya. Homeostasis must be maintained while cellular struc-This bears testimony to an old evolutionary principle. In tures are continually rebuilt, in particular during developfact, contrary to organelles such as the lysosome, selfment or in response to external stimuli. Proteins miscompartmentalizing molecular devices offer far greater folded as a consequence of mutations or ensuing from flexibility: when equipped with the appropriate localizaheat or oxidative stress must be scavenged because tion signals, they can be deployed to different cellular they are prone to aggregation. Beyond these more munlocations in the cytosol or in the nucleus, wherever their dane "housekeeping" functions, protein degradation action is needed. The advances made in recent years provides a means to terminate the lifespan of many in understanding the structure of the proteasome and its regulatory proteins at distinct times; amongst them are mechanism of action has helped to shape the concept of cyclins, transcription factors, and components of signal self-compartmentalization, and the proteasome became transduction pathways (for reviews, see Coux et al., the paradigm of this form of regulation. 1996; Hilt and Wolf, 1996;Varshavsky, 1997). Moreover, the immune system relies on the availability of immuno-The 20S Proteasome: Core of the competent peptides generated by the degradation of Proteolytic Machinery foreign antigens (for reviews, see Goldberg et al., 1995; 20S Proteasomes Are Found in All Three Heemels and Ploegh, 1995).Domains of Life However, since protein degradation is also a hazard,The first description of a "cylinder-shaped" complex it must be subject to spatial and temporal control in with proteasome-like features dates back to the late order to prevent the destruction of proteins not destined sixties. The plethora of names given to it subsequently for degradation. A basic stratagem in controlling protein is a reflection of the problems that were encountered degradation is compartmentalization, that is, the conover a period of two decades in trying to define its finement of the proteolytic action to sites that can only biochemical properties and cellular functions. Enzymobe accessed by proteins displaying some sort of degralogical studies revealed an array of distinct proteolytic dation signal. Such a compartment can be an organelle activities and led to a consensus name, "multicatalytic delimited by a membrane, as in the case of the lysosome.proteinase" (Dahlmann et al., 1988). ...

Proteasomes reach their mature active state via a complex cascade of folding, assembly and processing events. The Rhodococcus proteasome offers a means to dissect the assembly pathway and to characterize intermediates; its four subunits (oil, tx,2, Pi, P2) assemble efficiently in vitro with any combination of a and p. Assembly studies with wild-type and N-terminally truncated P-subunits in conjunction with refolding studies allowed to define the role of the propeptide which is two-fold: It supports the initial folding of the P-subunits and it promotes the maturation of the holoproteasomes.

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The 20S proteasome, isolated from the nocardioform actinomycete Rhodococcus erythropotts strain NI86/21, is built from two α-type and two ß-type subunits. In order to probe the subunit topology, we have set up an expression system which allows coexpression of the genes encoding the a-and ß-subunits in all possible combinations. The four respective constructs obtained yielded fully assembled and proteolytically active proteasomes. Biochemical, kinetic and electron microscopy analysis allow us to rule out several of the models which were originally envisaged for the subunit topology of the Rhodococcus proteasome. The experiments further indicate that the assembly pathways of the Rhodococcus and of the Thermoplasma proteasome differ in some important details.

Proteasomes are large, multisubunit proteases with highly conserved structures. The 26S proteasome of eukaryotes is an ATP-dependent enzyme of about 2 MDa, which acts as the central protease of the ubiquitin-dependent pathway of protein degradation. The core of the 26S complex is formed by the 20S proteasome, an ATP-independent, barrel-shaped protease of about 700 kDa, which has also been detected in archaebacteria and, more recently, in eubacteria. Currently, the distribution of 20S proteasomes in eubacteria appears limited to the actinomycetes, while most other eubacteria contain a related complex of simpler structure.

The 20 S proteasome, found in eukaryotes and in the archaebacterium Thermoplasma acldophilum, forms the proteolytic core of the 26 S proteasome which is the central protease of the non-lysosomal protein degradation pathway. Inhibitor studies have indicated that the 20 S proteasome may be an unusual type of cysteine or serine protease and a recent study of the Thermoplasma [3 subunit has indicated that it carries the proteolytic activity. We have attempted to obtain information on the nature of the active site by mutating the only cysteine, both histidines and two completely conserved aspartates in the archaebacterial complex as well as all serines of the/3 subunit, without decreasing the catalytic activity of the enzyme to any significant extent. Indeed, mutation of the conserved aspartate in the/3 subunit increased the activity of the proteasome threefold. We conclude that the proteasome is not a cysteine or serine protease.

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Viele Erkenntnisse über das Geschehen in einer Zelle vermitteln uns das Bild eines Fließgleichgewichts, das es der Zelle erlaubt, sich flexibel auf änderungen der Umwelt einzustellen. Während dieses Prinzip für Stoffwechselprozesse schon lange akzeptiertes Lehrbuchwissen darstellt, haben Untersuchungen zum Proteinabbau dieses Dogma nun auch auf proteolytische Prozesse erweitert. Die meisten zelluläen Proteine werden ständig neu gebildet und ebenso auch wieder abgebaut. Hierbei können sich ihre individuellen Halbwertszeiten je nach Funktion der Proteine stark voneinander unterscheiden. So besitzen Enzyme, die wichtige metabolische Kontrollfunktionen in der Zelle ausüben, Halbwertszeiten im Bereich von Minuten, während andere Proteine über Monate stabil vorliegen können. Der gezielte Proteinabbau stellt für die Zelle ein wirkungsvolles Kontrollinstrument dar, um die Konzentrationen wichtiger regulatorischer Proteine schnell und irreversibel zu senken. Auf diese Weise kann sich die Zelle zudem von abnormalen, wie zum Beispiel falsch gefalteten oder mutierten, und damit funktionsunfähigen Proteinen befreien, deren Ansammlung sie auf längere Sicht hin schäadigen würde. überdies werden wertvolle Aminosäurebausteine im Sinne eines effektiven Recyclings wieder zur Synthese neuer Proteine eingesetzt. Da die Proteinbiosynthese ein energieaufwendiger Prozeß ist, muß der regulierend eingesetzte Proteinabbau einer strikten Kontrolle unterliegen. Wir beginnen gerade zu verstehen, wie die Zelle das Kunststück fertigbringt, aus der großen Vielzahl aller zelluläen Proteine mit hoher Präzision diejenigen auszuwählen, die zu einem bestimmten Zeitpunkt abgebaut werden sollen.