Characterization of the protein dimerization domain responsible for assembly of functional selenodeiodinases
Molecular Endocrinology Laboratory; Department of Physiology; Program in Molecular Medicine
Amino Acid Sequence; Animals; Base Sequence; COS Cells; Dimerization; Humans; Hydrogen-Ion Concentration; Iodide Peroxidase; Molecular Sequence Data; Protein Processing, Post-Translational; Rats; Substrate Specificity
Life Sciences | Medicine and Health Sciences
Thyroid hormone metabolism is catalyzed by a small family of selenoenzymes. Type I deiodinase (D1) is the best characterized family member and is an integral membrane protein composed of two 27-kDa subunits that assemble to a functional holoenzyme after translation. To characterize the protein domain(s) responsible for this post-translational assembly event, we used deletion/truncation analysis coupled with immune depletion assays to map the dimerization domain of D1. The results of our studies show that a highly conserved sequence of 16 amino acids in the C-terminal half of the D1 subunit, -D148FL-YI-EAH-DGW163-, serves as the dimerization domain. Based on the high conservation of this domain, we synthesized a novel bait peptide-green fluorescent protein fusion probe (DDD(GFP)) to examine holoenzyme assembly of other family members. Overexpression of either the DDD(GFP) or an inert D1 subunit (M4) into SeD2 (accession number U53505)-expressing C6 cells specifically led to the loss of >90% of the catalytic activity. Catalytically inactive D2 heterodimers composed of SeD2: DDD(GFP) subunits were rescued by specific immune precipitation with anti-SeD2 IgG, suggesting that SeD2 requires two functional subunits to assemble a catalytically active holoenzyme. These findings identify and characterize the essential dimerization domain responsible for post-translational assembly of selenodeiodinases and show that family members can intermingle through this highly conserved protein domain.
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Citation: J Biol Chem. 2005 Mar 25;280(12):11093-100. Epub 2005 Jan 18. Link to article on publisher's site