|Ph.D Student||Rosenzweig Rina|
|Subject||Pinpointing Molecular Interactions with and within the Base|
Subcomplex of the 26S Proteasome
|Department||Department of Biology||Supervisor||Professor Michael Glickman|
|Full Thesis text|
The 26S proteasome is a multi-subunit, compartmentalized complex responsible for degradation of the vast majority of cellular proteins. It is comprised of two main components: the proteolytic 20S particle (CP), and the 19S regulatory particle (RP), the latter of which can be further separated into two stable subcomplexes: base and lid. The 19S RP binds ubiquitinated substrates, and subsequently de-ubiquitinates, unfolds and translocates them through an entry pore into the catalytic chamber of the 20S for degradation. While these functions are well recognized, little is known about the fine structure of the 19S or the delegation of tasks between its subunits. By using various biochemical purification techniques to shed layer after layer of peripheral subunits we have unearthed a novel functional unit within the base subcomplex of the 19S RP. We have also confirmed this observation by means of the opposite approach: using isolated subunits as bricks and cross linking reagents as mortar, we have partially rebuilt a synthetic regulatory complex from the bottom up on the surface of the 20S. In this manner, we have managed to expose the most rudimentary form of the proteasome regulatory particle in eukaryotes. Furthermore, breaking apart the 19S allowed us to use high resolution AFM to obtain first ever single particle images of individual components within the 19S in interaction with the 20S. This novel functional unit is composed of two concentric α-helical toroids, Rpn1 and Rpn2, which extend the proteolytic channel into the 20S. Rpn2 sits atop the cylindrical 20S, its inner cavity aligned with the entry pore , while the similarly structured Rpn1 stacks onto Rpn2 and serves as a docking site for substrate-recruitment factors. Together, Rpn1 and Rpn2 physically link the site of substrate recruitment and the site of proteolysis. The regulatory particle ATPases encircle Rpn1 and Rpn2, covering the remainder of the 20S-surface. We also found that Rpn1-Rpn2 participate in direct and indirect substrate recognition. Moreover, Rpn1-Rpn2 form a contiguous channel with the 20S and by the very act of binding, statically open the 20S channel thereby facilitating entry of small peptides without the need for energy derived from ATP hydrolysis.
By combining our own observations with pre-existing data, we propose a detailed model describing substrate trajectory from the site of recruitment to the site of proteolysis, along with the factors that are involved in every step of the way.