A novel component of the ubiquitination system, called NOSA, is essential for cellular differentiation in Dictyostelium discoideum. Disruption of nosA does not affect the growth rate but causes an arrest in development after the cells have aggregated. nosA contains seven exons and codes for a developmentally regulated 3.5-kb mRNA. The 125-kDa NOSA protein is present in the cytosol at constant levels during growth and development. The C-terminal region of NOSA has homology with ubiquitin fusion degradation protein-2 (UFD2) of Saccharomyces cerevisiae and putative homologs in Caenorhabditis elegans and humans. UFD2 is involved in the ubiquitin-mediated degradation of model substrates in which ubiquitin forms part of the translation product, but ufd2 mutants have no detected phenotype. In accord with the homology to UFD2, we found differences in the ubiquitination patterns between nosA mutants and their parental cell line. While general in vivo and in vitro ubiquitination is minimally affected, ubiquitination of individual proteins is altered throughout growth and development in nosA mutants. These findings suggest that events involving ubiquitination are critical for progression through the aggregate stage of the Dictyostelium life cycle.Protein levels and activities are regulated by differential gene expression, translational regulation, and post-translational modification. One such modification is the attachment of the small 8-kDa ubiquitin protein to a specific set of substrates, which targets these proteins to proteasomes for degradation. Although the ubiquitin pathway has the capacity to degrade almost every protein, it is a benign component of the cytoplasm and the nucleus. The protein degradation cascade begins when ubiquitin is attached to ubiquitin-activating enzymes of the E1 1 class through a high energy thioester bond. Activated ubiquitin is then transferred to substrates by ubiquitin-conjugating enzymes of the E2 class. The carboxyl-terminal glycine of ubiquitin forms a covalent bond with the ⑀-NH 2 group of a lysine in the target protein. In some cases, the conjugation of ubiquitin to proteins also requires the E3 ubiquitin ligases, which form complexes with specific E2 conjugating enzymes and the substrate, to confer specificity. Subsequent cycles result in the formation of multimeric ubiquitin chains on the target protein that are recognized by the 19 S cap of the proteasome. The multiubiquitin chain is removed by ubiquitinspecific hydrolases before the protein is unfolded and enters the 20 S proteasome for degradation (see Ref.