A role for the proteasome alpha2 subunit N-tail in substrate processing

The proteolytic active sites of the 26S proteasome are sequestered within the catalytic chamber of its 20S core particle (CP). Access to this chamber is through a narrow channel defined by the outer subunits. Free 20S CP is found in a latent state in which the N-termini of neighboring subunits form a gate blocking access to the channel. Entry of substrates can be facilitated by the attachment of the activators or regulatory particles, which can rearrange the blocking subunit N-terminal residues. In order to determine the specific physiological role of individual elements working in concert within the gate, we constructed a set of truncations or single-site mutations in each of the participating N-terminal tails. We report herein that whereas only a few N-termini are important for maintaining a closed gate, all seven N-termini participate in the open gate. Specifically, an invariant tyrosine (Y) in each subunit forms a hydrogen bond with a conserved aspartate (D) in the N-terminal tail of its counterclockwise neighbor, with the exception of the 1-2 pair leaving a gap in the ring circumference. A third residue (X) of this YD(X) motif aligns the open channel; specifically, the phenylalanine (F) at this position of the 2 subunit comes in direct contact with the translocating substrate. Consequently, deletion of this 2 N-terminal tail slows down proteolysis despite the appearance of an open gate state. We conclude that the YD(X) motif in N-terminal tail of subunits plays an important role in gating the proteasome and in processing the substrate.