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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, -D148ASFL-YI-EAHDGWM163-, serves as the dimerization domain. Based on the high conservation of this domain, we synthesized a novel bait peptide-green fluorescent protein fusion probe (DDDGFP) to examine holoenzyme assembly of other family members. Overexpression of either the DDDGFP 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: DDDGFP 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. [ABSTRACT FROM AUTHOR]

Type I deiodinase is the best characterized member of a small family of selenoenzymes catalyzing the bioactivation and disposal of thyroid hormone. This enzyme is an integral membrane protein composed of two 27-kDa subunits that assemble into a functional enzyme after translation using a highly conserved sequence of 16 amino acids in the C-terminal half of the polypeptide, 148DFLXXYIXEAHXXDGW163. In this study, we used alanine scanning mutagenesis to identify the key residues in this domain required for holoenzyme assembly. Overexpression of sequential alanine-substituted mutants of a dimerization domain-green fluorescent protein fusion showed that sequence 152IYI154 was required for type I enzyme assembly and that a catalytically active monomer was generated by a single I152A substitution. Overexpression of the sequential alanine-substituted dimerization domain mutants in type II selenodeiodinase-expressing cells showed that five residues (153FLIVY157) at the beginning and three residues (164SDG166) at the end of this region were required for the assembly of the type II enzyme. In vitro binding analysis revealed a free energy of association of -60 ± 5 kJ/mol for the noncovalent interaction between dimerization domain monomers. These data identify and characterize the essential residues in the dimerization domain that are responsible for the post-translational assembly of selenodeiodinases. [ABSTRACT FROM AUTHOR]