The ubiquitin-like protein Smt3p is activated for conjugation to other proteins by an Aos1p/Uba2p heterodimer

Abstract

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.

Stimulation of in vitro sumoylation by Slx5–Slx8: Evidence for a functional interaction with the SUMO pathway

“…The same procedure was used to purify the following point mutants as single subunits: Slx5-7 (C556S), Slx5-8 (C556S, H558A, C561S), Slx8-2 (C221S), Slx8-3 (C221S, H223A, C226S). The sumoylation enzymes His6-Aos1/Uba2, His6-Ubc9, and HF-Smt3 were purified from plasmids generously provided by Erica Johnson using published methods [37,38].…”

Drosophila Smt3 negatively regulates JNK signaling through sequestering Hipk in the nucleus

“…In mammalian cells, there are three SUMO genes, whereas only a single gene, smt3, exists in Drosophila (Huang et al, 1998;Johnson et al, 1997;Su and Li, 2002), making Drosophila a useful experimental system in which to study the biological functions of sumoylation. Several studies in Drosophila have suggested different functions of sumoylation, including in the regulation of cell signaling during development and ecdysteroid biosynthesis, but their underlying mechanisms remain largely unclear (Miles et al, 2008;Nie et al, 2009;Talamillo et al, 2008).…”

SUMO-1 Modification Alters ADAR1 Editing Activity

“…To confirm that the detection of GFP-SUMO-1 in the nucleolus corresponds to the presence of modified proteins, HeLa cells were transfected with GFP-SUMO-1⌬6. This mutant form of SUMO-1 lacks the C-terminal Gly-Gly motif and therefore cannot be conjugated to substrates (Johnson et al, 1997). Western blot analysis using antibodies against GFP shows that GFP-SUMO-1⌬6 is detected as a single band of the expected size ( Figure 1B, arrow), whereas full-length GFP-SUMO-1 is detected as a high-molecular-weight smear of conjugated products, indicating that this GFP fusion protein is effectively conjugated.…”