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Sumoylation of Ubc9 is required for the conjugating activity of UBC9
Author(s) -
Hwang Jaulang,
Yang Meiluen,
Lee JeiChen,
Chen Joanne,
Ko ChengYao
Publication year - 2007
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.21.6.a1019-a
Subject(s) - sumo protein , dna ligase , chemistry , ubiquitin ligase , cysteine , lysine , enzyme , thioester , ubiquitin , biochemistry , sumo enzymes , ubiquitin conjugating enzyme , amino acid , gene
Sumoylation involves an enzymatic cascade that resembles ubiquitination. The activating enzyme (SAE1/SAE2) and the conjugating enzyme (Ubc9) are sufficient for sumoylation of SUMO‐targeted proteins in vitro . In contrast to ubiquitination, sumoylation does not require E3 ligase in vitro , although specific SUMO E3 ligase has been reported to augment the sumoylation efficiency in vivo . A question was then raised whether the SUMO E3 ligase activity was compensated with other mechanisms in vitro . Our previous study (J. B. C. 281, 8264–8274) observed that there are two types of chemical bonds on the SUMO1 conjugated to Ubc9. One type of chemical bond is the thioester bond (sensitive to reduction by DTT) which is due to SUMO1 conjugation to the catalytic active cysteine residue of Ubc9, while the other type is the isopeptide bond (insensitive to reduction by DTT) which is counted as sumoylation of Ubc9. This study was taken efforts to determine which amino acid residue of Ubc9 was used for isopeptide bond formation and to ask whether there is a biological function of Ubc9 sumoylation. Mutagenetic analysis indicated the Lysine 65 of Ubc9, is the major residue for isopeptide bond formation. Interestingly, arginine substitution at lysine 65 of Ubc9 appears to abolish the conjugating activity of Ubc9 without affecting thioester bond formation between SUMO1 and cysteine 93 of Ubc9. This result indicates that sumoylation of Ubc9 is required for the conjugating activity of Ubc9. We also observed that the addition of GST‐RanBP2ΔFG possessing E3 ligase activity can partially restore conjugating enzyme activity of mutated Ubc9. This observation suggests that sumoylation of Ubc9 possibly involve in the transfer of SUMO1 or polySUMO1 moiety to substrate.