Protein Translocation Channels in the Proteasome and Other Proteases

Recently, two other intracellular proteases, the Gal6 protease of S. cerevisiae and the tricorn protease of T. acidophilum, have been likened to the proteasome in their structural organization (Figure 3Figure 3A and Figure 3Figure 3B). In the case of the hexameric protease Gal6, the crystal structure clearly shows a narrow (22 A) axial channel leading to a cavity containing the active sites (Joshua-Tor et al. 1995xJoshua-Tor, L, Xu, H.E, Johnston, S.A, and Rees, D.C. Science. 1995; 269: 945–950Crossref | PubMedSee all ReferencesJoshua-Tor et al. 1995). Tricorn similarly exists as a hexamer but is also capable of further assembly into a remarkable capsid-like structure of approximately 50 nm in diameter (Tamura et al. 1996xTamura, T, Tamura, N, Cejka, Z, Hegerl, R.H, Lottspeich, F, and Baumeister, W. Science. 1996; 274: 1385–1389Crossref | PubMed | Scopus (94)See all ReferencesTamura et al. 1996). Tricorn can also assemble with additional activating factors that themselves have peptidase activity (Tamura et al. 1996xTamura, T, Tamura, N, Cejka, Z, Hegerl, R.H, Lottspeich, F, and Baumeister, W. Science. 1996; 274: 1385–1389Crossref | PubMed | Scopus (94)See all ReferencesTamura et al. 1996). However, these factors do not have ATPase activity and have not been shown to cap the tricorn channel. Thus, the similarity of tricorn (and Gal6) to the proteasome may not extend to the regulatory complex. If ATP-dependent protein unfolding is a prerequisite for translocating protein substrates into the lumen, how could these proteases function? We suggest that there are two distinct classes of compartmentalized cytoplasmic proteases, ATP-dependent and ATP-independent. The latter class may be specialized in the hydrolysis of small molecules that can freely permeate the channel. For these enzymes, active site sequestration may enhance specificity by ensuring that they act on peptides rather than proteins.Figure 3Cut-Away Views of ATP-Independent, Proteasome-Like ComplexesCompiled with assistance from T. Tamura, W. Baumeister, and N. Straeter.(A) The tricorn protease. Note that in (A) and (B) the two-fold symmetry of the image results from the placement of the section plane, which is not a symmetry plane.(B) S. cerevisiae Gal6.(C) Bovine leucine aminopeptidase.View Large Image | View Hi-Res Image | Download PowerPoint SlideIs active site compartmentalization a feature of still other cytoplasmic peptidases? What may have been the first compartmentalized protease to be described is actually an exopeptidase (Burley et al. 1990xBurley, S.K, David, P.R, Taylor, A, and Lipscomb, W.N. Proc. Natl. Acad. Sci. USA. 1990; 87: 6878–6882Crossref | PubMedSee all ReferencesBurley et al. 1990). This passed largely unnoticed because it was reported before the proteasome structure had highlighted the significance of compartmentalization. Leucine aminopeptidase (LAP) is a hexameric enzyme with a three-membered ring structure, like Gal6 and tricorn (Table 1Table 1). LAP has six twisted channels that branch off from the equatorial plane (Figure 3Figure 3C). Because it is induced by γ-interferon, like certain proteasome subunits, LAP may play a role in antigen presentation, perhaps acting to trim peptides released from the proteasome itself.In summary, active sites have been sequestered in a large and otherwise unrelated set of cytoplasmic proteases, a shared feature that apparently arose through convergent evolution. However, the existence of ATP-independent peptidases with compartmentalized structures suggests that such enzymes may have supplied evolutionary precursors for the multicomponent ATP-dependent proteases that now mediate most selective degradation of protein substrates. The channels may have served originally as protein-excluding molecular sieves but became sites for highly selective protein translocation when coupled to ATP-hydrolyzing regulatory complexes. With the development of such regulatory complexes, substrate recognition was displaced from the proteolytically active subunits to the ATPases. In eukaryotes, the major specificity factors for degradation are displaced still further with the development of the ubiquitin-conjugation machinery, which can function independently of the proteasome. Through such steps, simple proteases have been replaced by elaborately regulated proteolytic pathways.*To whom correspondence should be addressed.