Topological Analysis of the Integral Membrane Protein, Type 1 Iodothyronine Deiodinase (D1) (*)
Topological Analysis of the Integral Membrane Protein, Type 1 Iodothyronine Deiodinase (D1) (*)
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DOI：
10.1074/jbc.270.20.12310
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发表时间：
1995-05
期刊：
影响因子：
--
通讯作者：
N. Toyoda;Marla J. Berry;J. Harney;P. Larsen
中科院分区：
文献类型：
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作者：
N. Toyoda;Marla J. Berry;J. Harney;P. Larsen
1型碘甲状腺原氨酸脱碘酶（D1）是一种微粒体硒酶，催化甲状腺素脱碘为3，5，3 ′-三碘甲状腺原氨酸。免疫印迹显示，内源性肝，肾，和瞬时表达的D1仍然在微粒体后，pH 11.5的处理。使用胰腺微粒体的体外翻译研究鉴定了具有胞质羧基末端催化部分的单个跨膜结构域。跨膜结构域位于位置11和12处的保守碱性氨基酸与位置34-39处的一组带电残基之间。其中残基2-25被删除的瞬时表达的D1蛋白是无活性的，并且不整合到膜中。活性没有恢复，取代这些残基与跨膜结构域的细胞色素P450或3型脱碘酶，尽管他们纳入膜。消除位置11和12处的正电荷使瞬时表达的蛋白质的量减少了70%，但形成的酶是催化正常的。在跨膜结构域中的Lys-27转化为Met或Glu后发现了类似的结果。我们得出结论，D1的氨基末端含有未切割的信号和停止转移序列的属性。此外，位置11、12和27处的带正电荷的残基是蛋白质的最佳形成所需的，但不是催化所需的。
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.