A novel ubiquitination factor, E4, is involved in multiubiquitin chain assembly. Proteins modified by multiubiquitin chains are the preferred substrates of the proteasome. Ubiquitination involves E1, E2, and E3 enzymes. The study shows that efficient multiubiquitination requires an additional factor, E4. E4 binds to ubiquitin moieties of preformed conjugates and catalyzes chain assembly with E1, E2, and E3. E4 defines a novel protein family, including UFD2 in yeast, NOSA in Dictyostelium, and human proteins. E4 activity is linked to cell survival under stress in yeast, indicating its role in eukaryotic proteolysis. E4 is essential for multiubiquitination of a model substrate, and its absence results in insufficient ubiquitin molecules for proteasomal degradation. E4 functions as a ubiquitin chain assembly factor, distinct from E1, E2, and E3. It interacts with CDC48, an AAA ATPase, and is involved in stress tolerance. E4 is functionally linked to stress tolerance in yeast, mediating degradation of stress-induced aberrant proteins. E4 is required for efficient multiubiquitination of Ubi-Proβgal in vivo. E4 is a novel ubiquitination factor that, like UFD2, is specifically required for multiubiquitin chain polymerization. E4 functions through its ubiquitin-binding property and may influence the linkage between individual ubiquitin molecules. E4 is involved in multiubiquitination in vivo, and its absence leads to reduced ubiquitin molecules on the substrate. E4 is essential for the degradation of UFD substrates in vivo. E4 is functionally linked to stress tolerance and likely mediates the degradation of stress-induced aberrant proteins. E4 is a novel ubiquitination factor that, like UFD2, is specifically required for multiubiquitin chain polymerization. E4 is involved in multiubiquitination in vivo, and its absence leads to reduced ubiquitin molecules on the substrate. E4 is essential for the degradation of UFD substrates in vivo. E4 is functionally linked to stress tolerance and likely mediates the degradation of stress-induced aberrant proteins.A novel ubiquitination factor, E4, is involved in multiubiquitin chain assembly. Proteins modified by multiubiquitin chains are the preferred substrates of the proteasome. Ubiquitination involves E1, E2, and E3 enzymes. The study shows that efficient multiubiquitination requires an additional factor, E4. E4 binds to ubiquitin moieties of preformed conjugates and catalyzes chain assembly with E1, E2, and E3. E4 defines a novel protein family, including UFD2 in yeast, NOSA in Dictyostelium, and human proteins. E4 activity is linked to cell survival under stress in yeast, indicating its role in eukaryotic proteolysis. E4 is essential for multiubiquitination of a model substrate, and its absence results in insufficient ubiquitin molecules for proteasomal degradation. E4 functions as a ubiquitin chain assembly factor, distinct from E1, E2, and E3. It interacts with CDC48, an AAA ATPase, and is involved in stress tolerance. E4 is functionally linked to stress tolerance in yeast, mediating degradation of stress-induced aberrant proteins. E4 is required for efficient multiubiquitination of Ubi-Proβgal in vivo. E4 is a novel ubiquitination factor that, like UFD2, is specifically required for multiubiquitin chain polymerization. E4 functions through its ubiquitin-binding property and may influence the linkage between individual ubiquitin molecules. E4 is involved in multiubiquitination in vivo, and its absence leads to reduced ubiquitin molecules on the substrate. E4 is essential for the degradation of UFD substrates in vivo. E4 is functionally linked to stress tolerance and likely mediates the degradation of stress-induced aberrant proteins. E4 is a novel ubiquitination factor that, like UFD2, is specifically required for multiubiquitin chain polymerization. E4 is involved in multiubiquitination in vivo, and its absence leads to reduced ubiquitin molecules on the substrate. E4 is essential for the degradation of UFD substrates in vivo. E4 is functionally linked to stress tolerance and likely mediates the degradation of stress-induced aberrant proteins.