The rapid turnover of spermidine/spermine N1-acetyltransferase (SSAT), a key enzyme in the regulation of polyamine levels, was found to be mediated via ubiquitination and the proteasomal system. SSAT degradation was blocked by the binding of polyamines or of the polyamine analog, N1,N12-bis(ethyl)spermine (BE-3-4-3), to the protein, providing a mechanism for the increase of SSAT activity in response to these agents. Site-directed mutagenesis indicated that a number of residues including arginine 19, cysteine 122, histidine 126, glutamic acid 152, arginine 155, and methionine 167 were needed for protection of SSAT by BE-3-4-3. These residues have previously been shown to reduce the affinity for the binding of polyamines to the SSAT protein, and these results indicate that the change in protein configuration brought about by this binding renders the protein resistant to proteasomal degradation. Mutations to alanines of residues arginine 7, cysteine 14, and lysine 141 also prevented the protection by BE-3-4-3, and these residues may be required for the formation of the protected conformation. The rapid degradation of SSAT required the carboxyl-terminal region of the protein, and the two terminal glutamic acid residues at positions 170 and 171 were found to be of critical importance. Truncation of the protein to remove these residues or the mutation of either of these acidic residues to glutamine completely abolished the rapid degradation of SSAT. The addition of two extra lysine residues at the carboxyl terminus or the conversion of the glutamic acids at positions 170 and 171 to lysines also prevented SSAT degradation by the proteasome. These results show the key role of the acidic residues at the carboxyl terminus of the protein in reacting with the proteasome. In contrast, mutation of lysine 166 to alanine, which extends the length of the acidic region in the carboxyl-terminal fragment of SSAT, actually increased the rate of degradation of SSAT without affecting its stabilization by BE-3-4-3. The binding of BE-3-4-3 or polyamines is therefore likely to change the configuration of the SSAT protein in a way that prevents the exposure of the carboxyl-terminal region of the ubiquitinated protein to the proteasome.