TOPOLOGICAL ANALYSIS OF THE INTEGRAL MEMBRANE-PROTEIN, TYPE-1 IODOTHYRONINE DEIODINASE (D1)

Type 1 iodothyronine deiodinase (D1) is a microsomal selenoenzyme whic h catalyzes deiodination of thyroxine to 3,5,3'-triiodothyronine. Immu noblotting showed that endogenous hepatic, renal, and transiently expr essed D1 remains in microsomes after pH 11.5 treatment. In vitro trans lation studies using pancreatic microsomes identified a single transme mbrane domain with a cytosolic carboxyl-terminal catalytic portion. Th e transmembrane domain is located between conserved basic amino acids at positions 11 and 12 and a group of charged residues at positions 34 -39. A transiently expressed D1 protein in which residues 2-25 were de leted was inactive and not integrated into membranes. Activity was not restored by replacing these residues with transmembrane domains from a cytochrome P450 or type 3 deiodinase enzyme despite their incorporat ion into membranes. Elimination of the positive charges at positions 1 1 and 12 reduced the amount of transiently expressed protein by 70%, b ut the enzyme formed was catalytically normal. Similar results were fo und after conversion of the Lys-27 in the transmembrane domain to Met or Glu. We conclude that the amino terminus of D1 contains uncleaved s ignal and stop transfer sequence properties. In addition, positively c harged residues at positions 11, 12, and 27 are required for optimal f ormation of the protein but not for catalysis.