The article investigates the recognition of polyubiquitin chains by the 26S proteasome, focusing on the role of tetraubiquitin (Ub4) as the minimum signal for efficient targeting. The authors used a structurally defined polyubiquitylated substrate, Ub5DHFR, to demonstrate that Ub4 is the shortest chain capable of binding to proteasomes with high affinity. They found that the affinity of Ub5DHFR for proteasomes depends nonlinearly on the length of the polyUb chain, with a transition occurring at chains of four Ubs. This suggests that the number of Ub4 units in a chain influences its signaling potential. The study also revealed that individual Ubs within Ub4 interact differently with proteasomal receptors, indicating that the Ub4 signal cannot be further subdivided. Additionally, the authors explored the properties of linear polyUb chains and found that they bind less tightly to proteasomes compared to K48-linked chains, suggesting that the conformation of the polyUb chain may influence its recognition by proteasomes. The results highlight the importance of substrate unfolding in the degradation process and suggest that extraproteasomal chaperones may be required for the efficient degradation of certain proteasome substrates.The article investigates the recognition of polyubiquitin chains by the 26S proteasome, focusing on the role of tetraubiquitin (Ub4) as the minimum signal for efficient targeting. The authors used a structurally defined polyubiquitylated substrate, Ub5DHFR, to demonstrate that Ub4 is the shortest chain capable of binding to proteasomes with high affinity. They found that the affinity of Ub5DHFR for proteasomes depends nonlinearly on the length of the polyUb chain, with a transition occurring at chains of four Ubs. This suggests that the number of Ub4 units in a chain influences its signaling potential. The study also revealed that individual Ubs within Ub4 interact differently with proteasomal receptors, indicating that the Ub4 signal cannot be further subdivided. Additionally, the authors explored the properties of linear polyUb chains and found that they bind less tightly to proteasomes compared to K48-linked chains, suggesting that the conformation of the polyUb chain may influence its recognition by proteasomes. The results highlight the importance of substrate unfolding in the degradation process and suggest that extraproteasomal chaperones may be required for the efficient degradation of certain proteasome substrates.