Altering the Stability of an Upstream Domain Affects the Proteasome's Ability to Unfold a Downstream Domain

The Ubiquitin-Proteasome System (UPS) is responsible for the bulk of protein degradation in eukaryotes. The 26S proteasome is the ATP-dependent protease component of the UPS that degrades misfolded, nonfunctional, and regulatory proteins. Substrates are targeted to the proteasome via polyubiquitination, followed by engagement, translocation, unfolding, and degradation. We are specifically interested in partial or incomplete degradation of substrates by the proteasome. Partial degradation can occur when the proteasome stalls on a difficult-to-unfold protein domain and then releases a portion of the protein as a stable fragment. Several "slippery substrates" have been identified, but the mechanisms that determine whether a substrate is fully or partially degraded remain unclear. We study the unfolding ability of the proteasome with a construct that contains an N-terminal degron, an easy-to-unfold barnase domain, and a harder-to-unfold DHFR domain. By measuring the extent to which DHFR is either fully degraded or released as a fragment, we can determine the proteasome's unfolding ability. Surprisingly, we have found that destabilizing the barnase domain via an L89G mutation (which has been reported to destabilize barnase by 7 kcal/mol) increases the ability of the proteasome to unfold the DHFR domain, even though no mutation or stability changes were made to DHFR. This result suggests that as the proteasome moves sequentially through a substrate, the stability of domains that it first encounters may affect the fate of downstream domains.