Tripeptidyl peptidase II (TPP II) is an exopeptidase of the subtilisin type of serine proteases that is thought to act downstream of the proteasome in the ubiquitin±proteasome pathway. Recently, a key role in a pathway parallel to the ubiquitin±proteasome pathway has been ascribed to TPP II, which forms a giant protease complex in mammalian cells. Here, we report the 900-fold puri®cation of TPP II from Drosophila eggs and demonstrate via cryo-electron microscopy that TPP II from Drosophila melanogaster also forms a giant protease complex. The presented three-dimensional reconstruction of the 57 3 27 nm TPP II complex at 3.3 nm resolution reveals that the 150 kDa subunits form a superstructure composed of two segmented and twisted strands. Each strand is 12.5 nm in width and composed of 11 segments that enclose a central channel. Keywords: cryo-electron microscopy/three-dimensional reconstruction/tripeptidyl peptidase II
IntroductionThe obvious necessity for compartmentalization of critical processes in the crowded cellular environment has led to the evolution of a number of large oligomeric protein complexes that bury their active sites in a secluded compartment. The paradigm of a self-compartmentalizing protease is the 20S proteasome, which, in eukaryotes, is capped bilaterally with the 19S regulatory complex to form the 26S proteasome Voges et al., 1999). This 2.4 MDa complex is the most downstream element of the ubiquitin±proteasome pathway, which controls diverse vital cellular functions (for reviews, see Coux et al., 1996;Zwickl et al., 2001;Glickman and Ciechanover, 2002). However, the potential of T-cell lymphoma cells to adapt to otherwise lethal concentrations of covalent inhibitors of the proteasome has led to an emerging consensus that there might be a parallel pathway allowing the cell to react more¯exibly in coping with its diverse physiological tasks, such as proteolytic house-keeping, major histocompatibility (MHC) class I-associated antigen presentation, apoptosis and the stress response (Glas et al., 1998). A key role in this alternative pathway is ascribed to tripeptidyl peptidase II (TPP II), which is upregulated in response to inhibition of the proteasome (Geier et al., 1999;Wang et al., 2000). In fact, endopeptidolytic activity has been demonstrated for this protease (Geier et al., 1999), although TPP II is primarily an exopeptidase of the subtilisin type of serine proteases and is otherwise thought to act downstream of the proteasome in the ubiquitin± proteasome pathway (Tomkinson, 1999), analogous to the role of tricorn protease in prokaryotes (Tamura et al., 1998). In addition to this soluble complex, a membranebound form of TPP II acting as a cholecystokinininactivating peptidase exists in rat cerebral cortex (Rose et al., 1996).Thus far, TPP II has only been found in eukaryotes. The mammalian variant has a molecular subunit mass of 138 kDa , whereas the insect, worm, plant and fungal form carry an additional insert in the C-terminal region and have a molecular mass of~150 kDa (Renn et a...
