Structure of a proteasome Pba1-Pba2 complex: implications for proteasome assembly, activation, and biological function.

The 20S proteasome is an essential, 28-subunit protease that sequesters proteolytic sites within a central chamber, thereby repressing substrate degradation until proteasome activators open the entrance/exit gate. Two established activators, Blm10 and PAN/19S, induce gate opening by binding to the pockets between proteasome α-subunits using C-terminal HbYX (hydrophobic-tyrosine-any residue) motifs. Equivalent HbYX motifs have been identified in Pba1 and Pba2, which function in proteasome assembly. Here, we demonstrate that Pba1-Pba2 proteins form a stable heterodimer that utilizes its HbYX motifs to bind mature 20S proteasomes in vitro and that the Pba1-Pba2 HbYX motifs are important for a physiological function of proteasomes, the maintenance of mitochondrial function. Other factors that contribute to proteasome assembly or function also act in the maintenance of mitochondrial function and display complex genetic interactions with one another, possibly revealing an unexpected pathway of mitochondrial regulation involving the Pba1-Pba2 proteasome interaction. Our determination of a proteasome Pba1-Pba2 crystal structure reveals a Pba1 HbYX interaction that is superimposable with those of known activators, a Pba2 HbYX interaction that is different from those reported previously, and a gate structure that is disrupted but not sufficiently open to allow entry of even small peptides. These findings extend understanding of proteasome interactions with HbYX motifs and suggest multiple roles for Pba1-Pba2 interactions throughout proteasome assembly and function.

Structural models for interactions between the 20S proteasome and its PAN/19S activators.

Proteasome activity is regulated by sequestration of its proteolytic centers in a barrel-shaped structure that limits substrate access. Substrates enter the proteasome by means of activator complexes that bind to the end rings of proteasome alpha subunits and induce opening of an axial entrance/exit pore. The PA26 activator binds in a pocket on the proteasome surface using main chain contacts of its C-terminal residues and uses an internal activation loop to trigger gate opening by repositioning the proteasome Pro-17 reverse turn. Subunits of the unrelated PAN/19S activators bind with their C termini in the same pockets but can induce proteasome gate opening entirely from interactions of their C-terminal peptides, which are reported to cause gate opening by inducing a rocking motion of proteasome alpha subunits rather than by directly contacting the Pro-17 turn. Here we report crystal structures and binding studies of proteasome complexes with PA26 constructs that display modified C-terminal residues, including those corresponding to PAN. These findings suggest that PA26 and PAN/19S C-terminal residues bind superimposably and that both classes of activator induce gate opening by using direct contacts to residues of the proteasome Pro-17 reverse turn. In the case of the PAN and 19S activators, a penultimate tyrosine/phenylalanine residue contacts the proteasome Gly-19 carbonyl oxygen to stabilize the open conformation.

Structure of the Blm10-20 S proteasome complex by cryo-electron microscopy. Insights into the mechanism of activation of mature yeast proteasomes.

The 20 S proteasome is regulated at multiple levels including association with endogenous activators. Two activators have been described for the yeast 20 S proteasome: the 19 S regulatory particle and the Blm10 protein. The sequence of Blm10 is 20% identical to the mammalian PA200 protein. Recent studies have shown that the sequences of Blm10 and PA200 each contain multiple HEAT-repeats and that each binds to the ends of mature proteasomes, suggesting a common structural and biochemical function. In order to advance structural studies, we have developed an efficient purification method that produces high yields of stoichiometric Blm10-mature yeast 20 S proteasome complexes and we constructed a three-dimensional (3D) model of the Blm10-20 S complex from cryo-electron microscopy images. This reconstruction shows that Blm10 binds in a defined orientation to both ends of the 20 S particle and contacts all the proteasome alpha subunits. Blm10 displays the solenoid folding predicted by the presence of multiple HEAT-like repeats and the axial gates on the alpha rings of the proteasome appear to be open in the complex. We also performed a genetic analysis in an effort to identify the physiological role of Blm10. These experiments, however, did not reveal a robust phenotype upon gene deletion, overexpression, or in a screen for synthetic effects. This leaves the physiological role of Blm10 unresolved, but challenges earlier findings of a role in DNA repair.