The 26 S proteasome is the central protease involved in ubiquitin-mediated protein degradation and fulfills vital regulatory functions in eukaryotes. The proteolytic core of the complex is the 20 S proteasome, a cylindrical particle with two outer rings each made of 7 different ␣-type subunits and two inner rings made of 7 different ␤-type subunits. In the archaebacterial 20 S proteasome ancestor proteolytically active sites reside in the 14 uniform ␤-subunits. Their N-terminal threonine residues, released by precursor processing, perform the nucleophilic attack for peptide bond hydrolysis. By directed mutational analysis of 20 S proteasomal ␤-type proteins of Saccharomyces cerevisiae, we identified three active site-carrying subunits responsible for different peptidolytic activities as follows: Pre3 for post-glutamyl hydrolyzing, Pup1 for trypsin-like, and Pre2 for chymotrypsin-like activity. Double mutants harboring only trypsin-like or chymotrypsin-like activity were viable. Mutation of two potentially active site threonine residues in the Pre4 subunit excluded its catalytic involvement in any of the three peptidase activities. The generation of different, incompletely processed forms of the Pre4 precursor in active site mutants suggested that maturation of non-active proteasomal ␤-type subunits is exerted by active subunits and occurs in the fully assembled particle. This trans-acting proteolytic activity might also account for processing intermediates of the active site mutated Pre2 subunit, which was unable to undergo autocatalytic maturation.The proteasome is a large multi-subunit proteinase complex found in the cytoplasm and nucleus of all eukaryotic cells examined so far. This "proteolytic organelle" fulfills vital cellular functions. As part of the ubiquitin-mediated protein degradation machinery, it is responsible not only for the elimination of misfolded proteins, including those derived from the lumen of the endoplasmic reticulum (1), it also controls a multitude of regulatory processes by removing unnecessary or even harmful metabolic enzymes and by balancing the levels of many regulatory proteins (for reviews see Refs. 2-4). Proteasomes exist as particles of 20 S and of 26 S. The 26 S complex of Ϸ2000 kDa is composed of the 20 S particle of Ϸ700 kDa as a proteolytic core unit and two regulatory 19 S caps that dock onto each side of the 20 S cylinder and confer ATP and ubiquitin dependence onto proteasomal protein degradation (2, 3, 5-7).A 20 S proteasome ancestor was isolated from the archaebacterium Thermoplasma acidophilum which exhibits an electron microscopic structure like the eukaryotic proteasome core but a much simpler subunit complexity. Extensive structural studies on this complex (8 -11) were completed by its x-ray crystallographic resolution (12). Two related subunits, ␣ and ␤, form a stack of four heptameric rings, whereby the two outer rings are composed of ␣-subunits and the two inner rings of ␤-subunits. Four narrow gates arranged along the cylinder axis give rise to three cavities...

Porin, also termed the voltage-dependent anion channel, is the most abundant protein of the mitochondrial outer membrane. The process of import and assembly of the protein is known to be dependent on the surface receptor Tom20, but the requirement for other mitochondrial proteins remains controversial. We have used mitochondria from Neurospora crassa and Saccharomyces cerevisiae to analyze the import pathway of porin. Import of porin into isolated mitochondria in which the outer membrane has been opened is inhibited despite similar levels of Tom20 as in intact mitochondria. A matrix-destined precursor and the porin precursor compete for the same translocation sites in both normal mitochondria and mitochondria whose surface receptors have been removed, suggesting that both precursors utilize the general import pore. Using an assay established to monitor the assembly of in vitro–imported porin into preexisting porin complexes we have shown that besides Tom20, the biogenesis of porin depends on the central receptor Tom22, as well as Tom5 and Tom7 of the general import pore complex (translocase of the outer mitochondrial membrane [TOM] core complex). The characterization of two new mutant alleles of the essential pore protein Tom40 demonstrates that the import of porin also requires a functional Tom40. Moreover, the porin precursor can be cross-linked to Tom20, Tom22, and Tom40 on its import pathway. We conclude that import of porin does not proceed through the action of Tom20 alone, but requires an intact outer membrane and involves at least four more subunits of the TOM machinery, including the general import pore.

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