A proteolytic pathway that recognizes ubiquitin as a degradation signal has been identified in yeast. This pathway, termed the UFD pathway, is responsible for degrading proteins that are fused to ubiquitin (Ub). The study identified five genes (UFD1, UFD2, UFD3, UFD4, and UFD5) involved in this pathway. UFD1, UFD2, and UFD4 encode previously unknown proteins of 40, 110, and 170 kDa, respectively. UFD3 plays a role in controlling the concentration of Ub in the cell, while UFD2 and UFD4 influence the formation and topology of multi-Ub chains linked to the fusion's Ub moiety. UFD5 is identical to the previously identified SON1 gene and is essential for both the UFD and N-end rule pathways. The study also shows that specific mutations in the Ub moiety significantly affect ubiquitination and degradation of Ub fusions. These findings suggest that Ub-Ub isopeptide bonds in substrate-linked multi-Ub chains involve both Lys48 and Lys29 of Ub, and that structurally different multi-Ub chains have distinct functions in Ub-dependent protein degradation. The UFD pathway is distinct from the N-end rule pathway, which degrades proteins with certain N-terminal residues. The study provides insights into the mechanisms of ubiquitination and proteolysis in eukaryotic cells.A proteolytic pathway that recognizes ubiquitin as a degradation signal has been identified in yeast. This pathway, termed the UFD pathway, is responsible for degrading proteins that are fused to ubiquitin (Ub). The study identified five genes (UFD1, UFD2, UFD3, UFD4, and UFD5) involved in this pathway. UFD1, UFD2, and UFD4 encode previously unknown proteins of 40, 110, and 170 kDa, respectively. UFD3 plays a role in controlling the concentration of Ub in the cell, while UFD2 and UFD4 influence the formation and topology of multi-Ub chains linked to the fusion's Ub moiety. UFD5 is identical to the previously identified SON1 gene and is essential for both the UFD and N-end rule pathways. The study also shows that specific mutations in the Ub moiety significantly affect ubiquitination and degradation of Ub fusions. These findings suggest that Ub-Ub isopeptide bonds in substrate-linked multi-Ub chains involve both Lys48 and Lys29 of Ub, and that structurally different multi-Ub chains have distinct functions in Ub-dependent protein degradation. The UFD pathway is distinct from the N-end rule pathway, which degrades proteins with certain N-terminal residues. The study provides insights into the mechanisms of ubiquitination and proteolysis in eukaryotic cells.