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J. Biol. Chem., Vol. 280, Issue 12, 11093-11100, March 25, 2005

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Characterization of the Protein Dimerization Domain Responsible for Assembly of Functional Selenodeiodinases^*

Jack L. Leonard, Gregory Simpson, and Deborah M. Leonard

From the Molecular Endocrinology Laboratory, Department of Cell and Molecular Physiology, University of Massachusetts Medical School, Worcester, Massachusetts 01655

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.

Received for publication, January 1, 2005

^* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: Molecular Endocrinology Laboratory, Dept. of Cell and Molecular Physiology, University of Massachusetts Medical School, 55 Lake Ave. N., Worcester, MA 01655. Tel.: 508-856-6687; Fax: 508-856-5997; E-mail: jack.leonard{at}umassmed.edu.

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