BIOCHEMICAL CHARACTERIZATIONS OF SUMO-1 CONJUGATION WITH PROTEINS IN XENOPUS EGG EXTRACTS

Abstract

SUMO-1 (Small Ubiquitin-related Modifer) is a small ubiquitin-related protein. Like ubiquitin, it can be covalently attached to other proteins via an isopeptide bond between its C-terminal glycine residue and lysine residues of substrate proteins. Conjugation of ubiquitin requires the activities of ubiquitin-activating El and ubiquitin-conjugating E2 enzymes. In an ATP-dependent process, the El enzyme forms a thioester with ubiquitin, which is transferred to the E2 enzyme. The E2 enzyme conjugates ubiquitin to the target substrates, in a process that also often requires an E3 ubiquitin ligase. The studies conducted for this thesis have investigated the biochemical similarities and differences between the ubiquitin and SUMO-I conjugation pathways. The results presented here show: First, SUMO-1 conjugation requires a diglycine peptide on the C-terminus of SUMO-I. Changing a conserved amino acid at the carboxyl-terminus of SUMO-I (Gly-97 to Ala-97) results in an abolition of conjugation with protein in Xeno pus extracts. An analogous mutation in ubiquitin does not abolish its conjugation, but does render ubiquitin insensitive to isopeptidases that catalyze its de-conjugation. Second, like the ubiquitin conjugation pathway, the SUMO-1 conjugation pathway is ATP-dependent in egg extracts, possibly reflecting a requirement for an El-like enzyme. Third, SUMO-1 conjugation is dependent upon Ubc9, a protein with homology to known ubiquitin E2 enzymes. Mutational analysis of Ubc9 suggests that its activity requires a cysteine residue that is conserved with ubiquitin E2 enzymes. Taken together, these studies suggest that the basic reaction mechanisms are conserved between he ubiquitin and SUMO-1 pathways, although the SUMO-1 pathway uses a distinct set of enzymes. Finally, an attempt has been made to identify novel substrates of the SUMO-1 conjugation pathway. The major conjugation product produced when exogenous GST-SUMO-1 was added to Xenopus egg extracts was purified and subjected to sequence analysis. The result show that it is a conjugated GST-SUMO-1 dimer, suggesting the possibility that SUMO-1 can be covalently conjugated into chains.