The study identifies tetraubiquitin as the minimum signal for efficient proteasomal targeting. Polyubiquitin chains linked through Lys48 are the principal signal for targeting substrates to the 26S proteasome. The research shows that tetraubiquitin is the minimum signal for efficient proteasomal targeting, and that the molecular basis of signal strength depends on chain length. The findings suggest that the higher-order conformation of the chain influences its signaling potential, and explain why a single ubiquitin is an inefficient proteasomal targeting signal. The study also shows that only a subset of potential interacting residues on the chain surface is important for recognition. The results indicate that the proteasome recognizes the polyubiquitin chain through a specific polymeric unit, which explains how a single ubiquitin can act as a functionally distinct signal. The properties of the substrates studied implicate substrate unfolding as a kinetically dominant step in the proteolysis of properly folded proteins, and suggest that extraproteasomal chaperones are required for efficient degradation of certain proteasome substrates. The study also shows that the proteasome binding of a linear polyubiquitin chain is influenced by the linkage in the chain, and that the chain's conformation can affect its signaling potential. The results suggest that the proteasome recognizes the polyubiquitin chain through a specific polymeric unit, and that the chain's conformation is important for its recognition by proteasomal receptors. The study also shows that the proteasome binding of a linear polyubiquitin chain is influenced by the linkage in the chain, and that the chain's conformation can affect its signaling potential. The results suggest that the proteasome recognizes the polyubiquitin chain through a specific polymeric unit, and that the chain's conformation is important for its recognition by proteasomal receptors. The study also shows that the proteasome binding of a linear polyubiquitin chain is influenced by the linkage in the chain, and that the chain's conformation can affect its signaling potential. The results suggest that the proteasome recognizes the polyubiquitin chain through a specific polymeric unit, and that the chain's conformation is important for its recognition by proteasomal receptors.The study identifies tetraubiquitin as the minimum signal for efficient proteasomal targeting. Polyubiquitin chains linked through Lys48 are the principal signal for targeting substrates to the 26S proteasome. The research shows that tetraubiquitin is the minimum signal for efficient proteasomal targeting, and that the molecular basis of signal strength depends on chain length. The findings suggest that the higher-order conformation of the chain influences its signaling potential, and explain why a single ubiquitin is an inefficient proteasomal targeting signal. The study also shows that only a subset of potential interacting residues on the chain surface is important for recognition. The results indicate that the proteasome recognizes the polyubiquitin chain through a specific polymeric unit, which explains how a single ubiquitin can act as a functionally distinct signal. The properties of the substrates studied implicate substrate unfolding as a kinetically dominant step in the proteolysis of properly folded proteins, and suggest that extraproteasomal chaperones are required for efficient degradation of certain proteasome substrates. The study also shows that the proteasome binding of a linear polyubiquitin chain is influenced by the linkage in the chain, and that the chain's conformation can affect its signaling potential. The results suggest that the proteasome recognizes the polyubiquitin chain through a specific polymeric unit, and that the chain's conformation is important for its recognition by proteasomal receptors. The study also shows that the proteasome binding of a linear polyubiquitin chain is influenced by the linkage in the chain, and that the chain's conformation can affect its signaling potential. The results suggest that the proteasome recognizes the polyubiquitin chain through a specific polymeric unit, and that the chain's conformation is important for its recognition by proteasomal receptors. The study also shows that the proteasome binding of a linear polyubiquitin chain is influenced by the linkage in the chain, and that the chain's conformation can affect its signaling potential. The results suggest that the proteasome recognizes the polyubiquitin chain through a specific polymeric unit, and that the chain's conformation is important for its recognition by proteasomal receptors.