Abstract
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.
Highlights
Thyroid hormone is essential for the normal growth and development of vertebrates
Gel filtration and density gradient centrifugation of detergent-soluble enzyme activity and/or affinity radiolabeled D1 showed that catalytic activity has an Mr of ϳ50,000 and that the ϳ27-kDa D1 polypeptide co-migrated with a complex, suggesting that D1 is a dimer of ϳ27-kDa subunits (9 –11)
The ability of an overexpressed catalytically inert D1 subunit to partner with Ͼ95% of the native p27 subunit(s) in LLC-PK1 cells [9] allowed us to use changes in catalytic activity as a measure of protein-protein interaction(s)
Summary
T3, 3Ј,3,5-triiodothyronine; rT3, 3Ј,5Ј,3-triiodothyronine; D1, D2, and D3, type I, II, and III iodothyronine deiodinase, respectively; GFP, green fluorescent protein; SeC, selenocysteine; TEMED, N,N,NЈ,NЈ-tetramethylethylenediamine; tDOC, taurodeoxycholate. Whether each subunit was catalytically functional or required post-translational assembly to become catalytically active was resolved by the finding that D1 dimers with only one functional subunit had ϳ50% of the activity of the wild-type enzyme [9, 12]. These hybrid holoenzymes had a molecular mass of 54 kDa and an S20,w of ϳ3.5 S, identical to that of the native D1 holoenzyme [9]. We determined the role of dimerization in the assembly of a catalytically active SeD2 enzyme using C6 cells that constitutively express the 30-kDa SeD2 subunit. For the D1 family member, catalytic activity is preserved when only one subunit is catalytically competent, whereas the D2 isoform required two fully functional subunits to be catalytically active
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