Topological analysis of the integral membrane protein, type 1 iodothyronine deiodinase (D1).
Type 1 iodothyronine deiodinase (D1) is a microsomal selenoenzyme which catalyzes deiodination of thyroxine to 3,5,3'-triiodothyronine. Immunoblotting showed that endogenous hepatic, renal, and transiently expressed D1 remains in microsomes after pH 11.5 treatment. In vitro translation studies using pancreatic microsomes identified a single transmembrane domain with a cytosolic carboxyl-terminal catalytic portion. The 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 deleted 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 incorporation into membranes. Elimination of the positive charges at positions 11 and 12 reduced the amount of transiently expressed protein by 70%, but the enzyme formed was catalytically normal. Similar results were found after conversion of the Lys-27 in the transmembrane domain to Met or Glu. We conclude that the amino terminus of D1 contains uncleaved signal and stop transfer sequence properties. In addition, positively charged residues at positions 11, 12, and 27 are required for optimal formation of the protein but not for catalysis.
Toyoda
N
N
Department of Medicine, Brigham and Women's Hospital, Boston, Massachusetts 02115, USA.
Berry
M J
MJ
Harney
J W
JW
Larsen
P R
PR
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D002474:Cell-Free System; D016384:Consensus Sequence; D018076:DNA, Complementary / Q000235:genetics; D004285:Dogs; D006801:Humans; D007453:Iodide Peroxidase / Q000737:chemistry* / Q000302:isolation & purification; D007668:Kidney / Q000201:enzymology; D008099:Liver / Q000201:enzymology; D008565:Membrane Proteins / Q000737:chemistry* / Q000302:isolation & purification; D008861:Microsomes / Q000201:enzymology; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009928:Organ Specificity; D010179:Pancreas / Q000201:enzymology; D014176:Protein Biosynthesis; D011487:Protein Conformation; D051381:Rats; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid
D018076:DNA, Complementary; D008565:Membrane Proteins; D007453:Iodide Peroxidase