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In situ structural studies of tripeptidyl peptidase II (TPPII) reveal spatial association with proteasomes

Significance Tripeptidyl peptidase II (TPPII) is a key player in protein catabolism. It is common among eukaryotes and believed to act downstream of the 26S proteasome. The giant exopeptidase shows a pronounced polymorphism in vitro; that is, its state of assembly is dependent on protein concentration. In situ structural studies by cryo-electron tomography with the Volta phase plate allow assessment of TPPII supramolecuar organization in a physiological setting. Moreover, cryo-electron tomography allows researchers to probe the spatial association of TPPII with proteasomes. This spatial association is consistent with biochemical data suggesting that TPPII acts as a postproteasomal protease. Our results suggest that the spatial association facilitates sequential protein breakdown by physical proximity. Tripeptidyl peptidase II (TPPII) is a eukaryotic protease acting downstream of the 26S proteasome; it removes tripeptides from the degradation products released by the proteasome. Structural studies in vitro have revealed the basic architecture of TPPII, a two-stranded linear polymer that assembles to form a spindle-shaped complex of ∼6 MDa. Dependent on protein concentration, TPPII has a distinct tendency for polymorphism. Therefore, its structure in vivo has remained unclear. To resolve this issue, we have scrutinized cryo-electron tomograms of rat hippocampal neurons for the occurrence and spatial distribution of TPPII by template matching. The quality of the tomograms recorded with the Volta phase plate enabled a detailed structural analysis of TPPII despite its low abundance. Two different assembly states (36-mers and 32-mers) coexist as well as occasional extended forms with longer strands. A distance analysis of the relative locations of TPPII and 26S proteasomes confirmed the visual impression that these two complexes spatially associate in agreement with TPPII’s role in postproteasomal degradation.