Identification, expression, and assay of an oxidation-specific ubiquitin ligase, HOIL-1.
The ubiquitin system plays important roles in the regulation of numerous cellular processes. It is well established that ubiquitin ligases (E3s) are key components in determining the specificity of the system and that the modification of substrates such as phosphorylation often plays a critical role in selective substrate recognition by E3s. Through studies analyzing iron-mediated degradation of iron regulatory protein 2 (IRP2), a central regulator of iron metabolism in mammalian cells, we have identified a RING finger protein, HOIL-1, as an ubiquitin ligase recognizing IRP2 through a signal created by heme-mediated oxidative modification of the protein. We have utilized several types of in vitro ubiquitination assays that detect IRP2 ubiquitination and a differential yeast two-hybrid screen in which yeast cells were cultured either in the presence or in the absence of oxygen to control the oxidation state of the bait in the cells in our studies. This chapter describes the detailed methods used for the identification and functional analysis of the HOIL-1 ligase.
Iwai
Kazuhiro
K
Department of Molecular Cell Biology, Graduate School of Medicine, Osaka City University, and Japan Science and Technology Agency, Osaka 545-8585, Japan.
Ishikawa
Haruto
H
Kirisako
Takayoshi
T
D000818:Animals; D016367:Baculoviridae; D045744:Cell Line, Tumor; D003001:Cloning, Molecular / Q000379:methods; D006367:HeLa Cells; D006418:Heme / Q000378:metabolism; D006801:Humans; D035942:Iron Regulatory Protein 2 / Q000235:genetics / Q000378:metabolism; D010084:Oxidation-Reduction; D018411:Spodoptera; D014157:Transcription Factors; D020798:Two-Hybrid System Techniques; D025801:Ubiquitin / Q000378:metabolism; D044763:Ubiquitin-Conjugating Enzymes / Q000235:genetics / Q000378:metabolism; D044767:Ubiquitin-Protein Ligases / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism*
D014157:Transcription Factors; D025801:Ubiquitin; D006418:Heme; D044763:Ubiquitin-Conjugating Enzymes; C504507:RBCK1 protein, human; D044767:Ubiquitin-Protein Ligases; D035942:Iron Regulatory Protein 2