Katherine Ferrell

Regulatory subunit interactions of the 26S proteasome, a complex problem

The 26S proteasome is the major non-lysosomal protease in eukaryotic cells. This multimeric enzyme is the integral component of the ubiquitin-mediated substrate degradation pathway. It consists of two subcomplexes, the 20S proteasome, which forms the proteolytic core, and the 19S regulator (or PA700), which confers ATP dependency and ubiquitinated substrate specificity on the enzyme. Recent biochemical and genetic studies have revealed many of the interactions between the 17 regulatory subunits, yielding an approximation of the 19S complex topology. Inspection of interactions of regulatory subunits with non-subunit proteins reveals patterns that suggest these interactions play a role in 26S proteasome regulation and localization.

Peptide sequencing identifies MSS1, a modulator of HIV Tat-mediated transactivation, as subunit 7 of the 26 S protease

Subunit 7 is an integral component of the human erythrocyte 26 S protease. Peptide sequence analysis reveals that 22 amino acids from the N‐terminus of subunit 7 correspond exactly to the N‐terminus of MSS1, a modulator of HIV gene expression. Additional internal peptides from subunit 7 obtained by CNBr cleavage also match 100% with the deduced amino acid sequence of MSS1. Based on the fact that directly sequenced peptides from subunit 7 are identical to more than 12% of the hypothetical translation product of MSS1, and the fact that the molecular weight of subunit 7 (49 kDa) corresponds to the predicted molecular weight of MSS1 (48,633 Da), we conclude that subunit 7 is MSS1.

Molecular cloning and expression of a multiubiquitin chain binding subunit of the human 26S protease

S5a is a subunit of the 26S protease that binds and presumably selects multiubiquitinated proteins for destruction. We recently identified an Arabidopsis protein, MBP1, that is physically, immunologically and biochemically similar to S5a from the human erythrocyte 26S protease. Based upon the MBP1 cDNA sequence we have now isolated a HeLa cell cDNA coding for human S5a. The HeLa cDNA sequence is highly similar to MBP1 and it encodes peptides obtained directly from human erythrocyte S5a. Moreover, expression of the isolated cDNA in E. coli results in a recombinant protein with an apparent molecular mass and multiubiquitin binding properties that match those of human S5a obtained from the purified 26S enzyme.

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 α 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 α 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 a Proteasome Pba1-Pba2 Complex: Implications for Proteasome Assembly, Activation, and Biological Function

Background: Pba1-Pba2 facilitates proteasome α-ring assembly. Results: Pba1-Pba2 binds mature proteasomes using C-terminal motifs and sequesters α-subunit N termini. It does not activate and is not degraded by isolated 20S proteasomes. Conclusion: Pba1-Pba2 is important for proteasome-dependent maintenance of mitochondrial function. The structure is consistent with multiple roles in proteasome assembly. Significance: Models of proteasome assembly and Pba1-Pba2 proteasome function are advanced. 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.

Molecular cloning and expression of subunit 12: a non-MCP and non-ATPase subunit of the 26 S protease

A cDNA encoding subunit 12 (S12) of human erythrocyte 26 S protease has been isolated, sequenced and expressed. The cDNA contains an open reading frame that encodes a 36.6 kDA protein 96% identical to mouse Mov‐34 and 67% identical to its Drosophila melanogaster homolog. Based on the high degree of sequence identity between human S12, mouse and Drosophila Mov‐34 proteins, we conclude that the Mov‐34 gene product is a component of the 26 S protease. Antibodies produced against two S12 fragments, Met1‐Tyr95 (S12f95) and Met1‐Leu205 (S12f205), react with S12 transferred to nitrocellulose from SDS‐PAGE. In contrast, after transfer from native gels, the epitope(s) recognized by anti‐S12f205 is exposed in the regulatory complex but appears to be masked when the regulatory complex associates with the multicatalytic protease.