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 ...