Abstract: The p50 subunit of NF-kappa B is generated by proteolytic processing of a 105-kDa precursor (p105) in yeast and mammalian cells. Here we show that yeast mutants in the ubiquitin-proteasome pathway inhibit or abolish p105 processing. Specifically, p105 processing is inhibited by a mutation in a 20 S proteasome subunit (pre1-1), by mutations in the ATPases located in the 19 S regulatory complexes of the proteasome (yta1, yta2/sug1, yta5, cim5), and by a mutation in a proteasome-associated isopeptidase (doa4).A ubiquitinated intermediate of the p105 processing reaction accumulates in some of these mutants, strongly suggesting that ubiquitination is required for processing. However, none of the ubiquitin conjugating enzyme mutants tested (ubc1, -2, -3, -4/5, -6/7, -8, -9, -10, -11) had an effect on p105 processing, suggesting that more than one of these enzymes is sufficient for p105 processing. Interestingly, a mutant "N-end rule" ligase does not adversely affect p105 processing, showing that the N-end rule pathway is not involved in degrading the C-terminal region of p105. Unexpectedly, we found that a glycine-rich region of p105 that is required for p105 processing in mammalian cells is not required for processing in yeast. Thus, p105 processing in both yeast and mammalian cells requires the ubiquitin-proteasome pathway, but the mechanisms of processing, while similar, are not identical.