RP - In vitro polyubiquitination assay (»Add to Infobox)

Wolf Dieter H.

Dieter H. Wolf's Group

An in vitro polyubiquitination assay of proteins is a powerful tool to prove the in vivo function of proteins in ubiquitination. The in vitro system allows to find components of the ubiquitination machinery. We have used the assay to prove ubiquitin ligase activity of a subunit of the Gid complex, Gid2/RMD5 (Santt et al., 2008). As shown in (Regelmann et al., 2003), GID2/RMD5 deletion prevents polyubiquitination of FBPase. Alignments of the Gid2/Rmd5 protein sequence with known RING-finger E3´s revealed that it bears a so-called degenerated RING-finger where Zn2+ coordination residues are missing (Santt et al., 2008). A complete cysteine and histidine pattern in a RING domain is not necessarily critical for the E3 function, as the U-box domain family shows (Ohi et al., 2003). The canonical RING domain encompasses eight residues coordinating two Zn cations. The first Zn2+ ion is coordinated by the first, second, fifth and sixth residue; the second by the remaining residues three, four, seven and eight (Fang and Weissman, 2004; Lorick et al., 1999). In Gid2 besides the first cysteine, the four residues coordinating the second Zn2+ ion are conserved, which strongly suggests that one Zn2+ is retained in this degenerated RING domain. This prompted us to suspect that Gid2 may bear an ubiquitin ligase activity.

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Wolf Dieter H. (Principal Investigator / Dieter H. Wolf's Group)

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