This study investigates the UFD pathway (Ub fusion degradation) in yeast, which is responsible for the rapid degradation of proteins bearing a nonremovable N-terminal ubiquitin (Ub) moiety. The UFD pathway involves several genes, including UFD1, UFD2, UFD3, UFD4, and UFD5, each playing distinct roles in the pathway. UFD3 controls the concentration of free Ub, while UFD4 and UFD2 influence the formation and topology of multi-Ub chains. UFD1 encodes a protein similar to E6AP, a human protein involved in Ub-dependent protein degradation. The study also examines the degradation of different Ub fusions, such as Ub-P-βgal and UbV76-V-βgal, and their mutants, to understand the specific functions of these genes. Overexpression of Ub can partially rescue the degradation defects in some ufd mutants, particularly ufd3, suggesting that the primary defect in this mutant is related to Ub concentration. The findings provide insights into the mechanisms of Ub-dependent protein degradation and the roles of specific genes in this process.This study investigates the UFD pathway (Ub fusion degradation) in yeast, which is responsible for the rapid degradation of proteins bearing a nonremovable N-terminal ubiquitin (Ub) moiety. The UFD pathway involves several genes, including UFD1, UFD2, UFD3, UFD4, and UFD5, each playing distinct roles in the pathway. UFD3 controls the concentration of free Ub, while UFD4 and UFD2 influence the formation and topology of multi-Ub chains. UFD1 encodes a protein similar to E6AP, a human protein involved in Ub-dependent protein degradation. The study also examines the degradation of different Ub fusions, such as Ub-P-βgal and UbV76-V-βgal, and their mutants, to understand the specific functions of these genes. Overexpression of Ub can partially rescue the degradation defects in some ufd mutants, particularly ufd3, suggesting that the primary defect in this mutant is related to Ub concentration. The findings provide insights into the mechanisms of Ub-dependent protein degradation and the roles of specific genes in this process.