Identification of ubiquitin ligase substrates by in vitro expression cloning

Nagi G. Ayad, Susannah Rankin, Danny Ooi, Michael Rape, Marc W. Kirschner

Research output: Contribution to journalReview articlepeer-review

19 Scopus citations


The number of identified E3 ubiquitin ligases has dramatically increased in recent years. However, the substrates targeted for degradation by these particular ligases have not been easily identified. One reason for the inability of matching substrates and ligases is the finding that E3 recognition elements in substrates are often poorly defined. This minimizes the likelihood that bioinformatic approaches will lead to the identification of E3 substrates. For example, the multi-subunit complex the anaphase promoting complex (APC) is an E3 that recognizes destruction boxes (RXXLXXXXD/N/E) or KEN motifs within substrates (Glotzer et al., 1991; Pfleger and Kirschner, 2000). However, many proteins that contain either a potential destruction or a KEN motif are not recognized by the APC in vitro or in vivo, suggesting that there are other, less well-defined characteristics of substrates that contribute to their ability to serve as APC substrates (Ayad, Rankin, and Kirschner, unpublished observations). Aside from bioinformatic approaches of identifying APC substrates, several groups have also attempted to use affinity techniques to discover novel APC substrates. This has not been widely successful, because many APC substrates are not abundant. Also, as is the case with many ligase-substrate interactions, the affinity of substrates for the APC is likely to be very low. All these considerations have motivated a search for other techniques to assist in identifying substrates of this particular E3 ligase. Here, we describe the use of in vitro expression cloning to identify novel APC substrates.

Original languageEnglish (US)
Article number28
Pages (from-to)404-414
Number of pages11
JournalMethods in enzymology
StatePublished - 2005
Externally publishedYes

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology


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