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Identification of an Arabidopsis WD‐Repeat Protein that Activates the Deubiquitinase UBP3 and Interacts with Two E3 Ubiquitin Ligases
Author(s) -
Baskerville Andrew T,
Donahue Janet,
Gillaspy Glenda,
Erickson Les
Publication year - 2017
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.31.1_supplement.lb102
Subject(s) - deubiquitinating enzyme , ubiquitin , arabidopsis , biology , proteases , ubiquitin ligase , f box protein , proteasome , microbiology and biotechnology , biochemistry , ubiquitin protein ligases , mutant , gene , enzyme
Ubiquitination is an essential, dynamic post‐translational modification that regulates a diverse set of cellular processes. Ligation of the small peptide ubiquitin (Ub) onto substrate proteins by E3 Ub ligases can change protein localization, activity or stability. The most common outcome for Ub‐tagged proteins is degradation via the 26S proteasome. Deubiquitinases (DUBs) reverse the effects of ubiquitination by removing Ub from tagged substrates. In animals, Ubiquitin‐specific protease (USP) 12 and USP46 are related DUBs that regulate key proteins in important growth and differentiation signaling pathways. USP12 and USP46 each form trimeric complexes with two WD‐repeat (WDR) proteins, WDR20 and WDR48. The WDR proteins activate the enzymatic abilities of the DUBs while potentially serving as substrate receptors. Numerous animal and fungal DUBs are bound and activated by specific WDR proteins. In Arabidopsis , Ub‐specific proteases 3 (AtUBP3) and 4 (AtUBP4) are related DUBs that have significant sequence similarities to animal USP12/46. Genetic studies have shown AtUBP3/4 are essential for proper pollen development and transmission. It is not yet known if WDR proteins bind and activate DUBs in plants. We have identified At2g37160 as a likely Arabidopsis WDR20 homolog based on amino acid sequence homology and the number and organization of WD motifs. We have shown At2g37160 (AtWDR20) interacts with both AtUBP3 and AtUBP4 in the yeast two‐hybrid system. Biochemical characterization using recombinant proteins shows AtWDR20 increases the DUB activity of AtUBP3 over nine‐fold. A yeast two‐hybrid screen of an Arabidopsis cDNA library using AtWDR20 as bait revealed interactions between AtWDR20 and two different E3 Ub ligases: Myb‐30 Interacting E3 Ub ligase (MIEL1) and CHY Zinc‐finger and Ring Protein 1 (CHYR1). Interactions between a DUB‐associated WDR protein and E3 Ub ligases is intriguing, and implies AtWDR20 may function on both sides of the Ub cycle or is itself a target of ubiquitination. In planta and in vitro analyses will further characterize the interactions between AtWDR20, AtUBP3, and these E3 ligases.