The ubiquitin-like protein Smt 3 p is activated for conjugation to other proteins by an Aos 1 p / Uba 2 p heterodimer mere-binding protein ( Meluh and Koshland , 1995 ) . Smt 3 p

mere-binding protein (Meluh and Koshland, 1995). Smt3p Erica S.Johnson1, Ingrid Schwienhorst2, is 48% identical to SUMO-1 and 17% identical to Ub. R. Jürgen Dohmen2 and Günter Blobel The 76-residue protein Ub is highly conserved, with Laboratory of Cell Biology, Howard Hughes Medical Institute, only three conservative changes between yeast and man. Rockefeller University, New York, NY 10021, USA and All known functions of Ub, many of which involve 2Institut für Mikrobiologie, Heinrich-Heine-Universität Düsseldorf, proteasome-dependent proteolysis, are mediated by its Universitätsstraße 1, Geb. 26.12, D-40225, Düsseldorf, Germany conjugation to other proteins (reviewed by Hochstrasser, 1Corresponding author 1996; King et al., 1996). A number of proteins including e-mail: johnsoe@rockvax.rockefeller. edu Smt3p and SUMO-1 contain Ub-like domains with much less sequence similarity to Ub than among genuine Ub SMT3 is an essential Saccharomyces cerevisiae gene homologs. Most of these, including the S.cerevisiae encoding a 11.5 kDa protein similar to the mammalian nucleotide excision repair protein Rad23p (Watkins et al., ubiquitin-like protein SUMO-1. We have found that 1993) and Dsk2p, involved in spindle pole body duplicSmt3p, like SUMO-1 and ubiquitin, can be attached ation (Biggins et al., 1996), do not become conjugated to to other proteins post-translationally and have characother proteins. The only Ub-like proteins other than terized the processes leading to the activation of the SUMO-1 shown to form conjugates are the interferonSmt3p C-terminus for conjugation. First, the SMT3 inducible protein UCRP (Loeb and Haas, 1992) and a translation product is cleaved endoproteolytically to baculovirus Ub variant (Haas et al., 1996). One distinexpose Gly98, the mature C-terminus. The presence of guishing feature of Ub-like proteins capable of conjugation Gly98 is critical for Smt3p’s abilities to be conjugated is likely to be a diglycine sequence corresponding to the to protein substrates and to complement the lethality C-terminal Gly75 and Gly76 of Ub, which is critical for of a smt3Δ strain. Smt3p undergoes ATP-dependent interactions between Ub and Ub-pathway enzymes. Like activation by a novel heterodimeric enzyme consisting SUMO-1, UCRP and the baculovirus Ub variant, Smt3p of Uba2p, a previously identified 71 kDa protein similar contains these glycine residues. to the C-terminus of ubiquitin-activating enzymes Post-translational attachment of Ub to other proteins is (E1s), and Aos1p (activation of Smt3p), a 40 kDa carried out by a multi-step pathway culminating in formprotein similar to the N-terminus of E1s. Experiments ation of an isopeptide bond between the C-terminal with conditional uba2 mutants showed that Uba2p is carboxyl group of Ub and the ε-amino group of a lysine required for Smt3p conjugation in vivo. Furthermore, side-chain in an acceptor protein. All Ub genes encode UBA2 and AOS1 are both essential genes, providing natural fusion proteins in which Ub’s C-terminus is linked additional evidence that they act in a distinct pathway to another copy of Ub, a small ribosomal protein or an whose role in cell viability is to conjugate Smt3p to oligopeptide (Özkaynak et al., 1987; Jentsch et al., 1991). other proteins. Ub fusions are rapidly cleaved by Ub-specific endo