UC Berkeley

The ubiquitin-proteasome system is largely responsible for the turnover of proteins in the

eukaryotic cell. The proteasome is a compartmental protease of the AAA+ family, and it

mechanically unfolds ubiquitinated substrates into a proteolytic chamber in an ATPdependent

manner for degradation. This mechanical unfolding is performed by the

proteasome base, but the AAA-motor can only function as a part of the larger proteasome

complex. In addition to unfolding, the proteasome must first be recognize substrates by

their attached ubiquitin chains and thread them into the motor. Ubiquitin modifications

must be removed by the deubiquitinase Rpn11. This dissertation focuses on the effect of

substoichiometric proteasome components in the assembly and function of the base. First,

this dissertation focuses on how substoichiometric proteasome components Ubp6 and

Rad23 affect proteasomal degradation through their interactions with the base.

Ubp6, a deubiquitinase, affects the conformational state of the proteasome through direct

interaction with the N-ring in the base in addition to interactions with base subunit Rpn1.

By enforcing a substrate-engaged conformation in the proteasome, ubiquitin-bound Ubp6

inhibits Rpn11, stimulates ATPase activity, and shuts down substrate turnover. Ubp6 acts

more slowly on ubiquitin-protein substrates than Rpn11 but displays higher activity towards

non-translocation dependent ubiquitin substrates. Rad23 also binds to Rpn1 and serves as a

ubiquitin receptor at the proteasome. Substrates delivered through Rad23 are

deubiquitinated and degraded more slowly than those delivered through the intrinsic

ubiquitin receptor, Rpn10. However, substrates delivered through either receptor are

deubiquitinated by Ubp6 at similar rates.

Finally, this dissertation examines the importance of four base-specific assembly factors in

heterologous expression of the base. These assembly factors, Nas6, Rpn14, Hsm3, and

Nas2, help to stabilize base subunits, which allows for assembly, but they are unable to

prevent the assembly of base subunits into incorrect complexes. In addition, other

components of the proteasome, Ubp6 and core, discriminate between properly and

improperly assembled base.