The following S. pombe strains were used in this study: csn5∆ (RDY 1712)(S6); Pcu1 myc13 (RDY 1568)(S7); csn5∆, Csn1 myc13 (RDY 2092)(S8); csn5∆, Csn2 myc13 (RDY 2093)(S8).

The growth-suppressive properties of p53 are controlled by posttranslational modifications and by regulation of its turnover rate. Here we show that p53 can be modified in vitro and in vivo by conjugation to the small ubiquitin-like protein SUMO-1. A lysine residue at amino acid position 386 of p53 is required for this previously undescribed modification, strongly suggesting that this lysine residue serves as the major attachment site for SUMO-1. Unlike ubiquitin, attachment of SUMO-1 does not appear to target proteins for rapid degradation but rather, has been proposed to change the ability of the modified protein to interact with other cellular proteins. Accordingly, we provide evidence that conjugation of SUMO-1 to wild-type p53 results in an increased transactivation ability of p53. We suggest that posttranslational modification of p53 by SUMO-1 conjugation provides a novel mechanism to regulate p53 activity.

The ubiquitin-like protein SMT3 from Saccharomyces cerevisiae and SUMO-1, its mammalian homolog, can be covalently attached to other proteins posttranslationally. Conjugation of ubiquitin requires the activities of ubiquitin-activating (E1) and -conjugating (E2) enzymes and proceeds via thioester-linked enzyme-ubiquitin intermediates. Herein we show that UBC9, one of the 13 different E2 enzymes from yeast, is required for SMT3 conjugation in vivo. Moreover, recombinant yeast and mammalian UBC9 enzymes were found to form thioester complexes with SMT3 and SUMO-1, respectively. This suggests that UBC9 functions as an E2 in a SMT3͞SUMO-1 conjugation pathway analogous to ubiquitinconjugating enzymes. The role of yeast UBC9 in cell cycle progression may thus be mediated through its SMT3 conjugation activity.Ubiquitin, a highly conserved protein of 76 amino acid residues, can be attached to other proteins posttranslationally (for reviews, see refs. 1-3). This reaction involves the formation of an isopeptide bond between the carboxyl-terminal glycine residue of ubiquitin and the -amino group of a lysine residue of an acceptor protein. All known functions of ubiquitin, including its role in selective protein degradation, are thought to be mediated through this reaction. Conjugation of ubiquitin proceeds via a reaction cascade involving ubiquitin-activating (E1) and ubiquitin-conjugating (E2) enzymes and, at least in some cases, ubiquitin-protein ligases (E3). The E1 enzyme hydrolyzes ATP and forms a high-energy thioester intermediate between a cysteine residue of its active site and the carboxyl terminus of ubiquitin. Ubiquitin is then passed on to E2 enzymes that form thioesters with ubiquitin in a similar fashion. Finally, ubiquitin is covalently attached to a substrate protein by the E2 enzymes or, alternatively, by E3 enzymes as indicated by the observation that at least some E3 enzymes are loaded with ubiquitin by E2 enzymes via thioester formation (4).The yeast Saccharomyces cerevisiae genome contains several homologous genes encoding putative E1 enzymes (UBA genes). UBA1 is essential for viability and encodes the activating enzyme required for ubiquitin conjugation (5). UBA2, also required for viability, encodes a smaller protein that bears a cysteine residue at a position similar to the active-site cysteine of UBA1 (6). A uba2 mutant, expressing a protein that lacks this cysteine, is inviable, suggesting that this conserved residue is involved in thioester formation as well. Surprisingly, however, purified UBA2 failed to form a thioester complex with ubiquitin, suggesting that it may function in a different pathway (6). Indeed, UBA2 was recently shown to cooperate with another protein termed AOS1 in the activation of SMT3, a protein of 98 amino acid residues with 17% sequence similarity to ubiquitin (7). Interestingly, AOS1, also encoded by an essential gene, and UBA2 show sequence similarity to the amino-and the carboxyl-terminal domains of the UBA1 protein, respectively. This suggests that upon heterodi...