To degrade or not to degrade : protein handling by the proteasome

Abstract

The proteasome, a large multi-subunit protease that degrades proteins, must be able to distinguish between substrates deemed for destruction and accessory proteins that should be spared. The shuttle factor Rad23 operates in close proximity to the proteasome in order to deliver proteins for their degradation. While its cargo is efficiently degraded, the shuttle factor itself remains unharmed. Rad23 is protected by its C-terminal ubiquitin-associated (UBA)-2 domain that has been identified to be a cisacting stabilization signal. A major aim of the work presented in this dissertation has been to decipher the mode of action responsible for this protective effect.

Another protein that is targeted to the proteasome but resists degradation was studied in Paper I. UBB+1 is an aberrant product of the ubiquitin precursor gene UBB and comprises ubiquitin and a 19 amino acid-long C-terminal extension. Even though UBB+1 structurally resembles a ubiquitin fusion degradation (UFD) substrate, a class of proteins that is targeted for degradation by a non-cleavable N-terminal ubiquitin moiety, it is poorly degraded. We found that designed UFD substrates with equally short C-terminal extensions were stable. Extending their C-termini to 25 amino acids converted them into short-lived proteins, suggesting that the proteasome requires an unstructured polypeptide of a minimum length from which unfolding of the substrate may be initiated.

In Paper II we further investigated the impact of unstructured polypeptide sequences on proteasomal degradation. We found that C-terminal initiation sites for proteasomal unfolding rendered the degradation of UFD substrates independent of the ubiquitin-binding chaperone Cdc48 and polyubiquitylation. This observation suggests that substrates bypassing the unfoldase activity of Cdc48 require an unstructured initiation site for their efficient degradation and implies that the chaperone complex acting upstream of the proteasome can be the primary determinant for the polyubiquitin-dependency of proteasomal degradation. Consistently, we found that the ubiquitin-like (UbL) domains of Rad23 and Dsk2, which interact with the proteasome in a polyubiquitin-independent manner, only sufficed to target proteins carrying initiation sites for degradation.

The impact of C-terminal unstructured polypeptide sequences on proteasomal degradation prompted us to investigate the role of structural integrity in UBA-mediated protection in Paper III. To this end, we used the C-terminal UBA domain of the human protein p62 and took advantage of naturally occurring mutant domains that are linked to Paget s disease of bone. We found that not only the wild-type UBA domain could delay degradation of reporter substrates in yeast but also mutant domains that were thermally stable but impaired in their ubiquitin binding. This suggests that the UBA-mediated protective effect depends rather on the structural integrity than ubiquitin binding capability.

Our finding that proteins targeted to the proteasome through a UbL domain require an unstructured initiation site for efficient degradation turned out to be of significance for understanding the molecular mechanism behind Rad23 protection and encouraged us to study in Paper IV whether the protective UBA domains interfered with unfolding. Strikingly, introduction of C-terminal unstructured polypeptides turned the shuttle factor Rad23 into an efficiently degraded proteasome substrate. Positioning the UBA2 domain C-terminally to an adjacent unstructured polypeptide inhibited degradation, whereas non-protective UBA domains were able to function as initiation sites themselves.

In summary, we provide evidence that the protective effect of UBA domains is mediated by preventing initiation of degradation by the proteasome. These molecular insights help explain how proteasomes decide which proteins to degrade and which proteins to spare and thus how shuttle factors can deliver substrates to the proteasome without themselves becoming subject to degradation.