The 26S Proteasome Switches between ATP-Dependent and -Independent Mechanisms in Response to Substrate Ubiquitination.

The ubiquitin-proteasome system is responsible for the bulk of protein degradation in eukaryotic cells. Proteins are generally targeted to the 26S proteasome through the attachment of polyubiquitin chains. Several proteins also contain ubiquitin-independent degrons (UbIDs) that allow for proteasomal targeting without the need for ubiquitination. Our laboratory previously showed that UbID substrates are less processively degraded than ubiquitinated substrates, but the mechanism underlying this difference remains unclear. We therefore designed two model substrates containing both a ubiquitination site and a UbID for a more direct comparison. We found UbID degradation to be overall less robust, with complete degradation only occurring with loosely folded substrates. UbID degradation was unaffected by the nonhydrolyzable ATP analog ATPγS, indicating that UbID degradation proceeds in an ATP-independent manner. Stabilizing substrates halted UbID degradation, indicating that the proteasome can only capture UbID substrates if they are already at least transiently unfolded, as confirmed using native-state proteolysis. The 26S proteasome therefore switches between ATP-independent weak degradation and ATP-dependent robust unfolding and degradation depending on whether or not the substrate is ubiquitinated.