The N-terminal anchor sequences of 11β-hydroxysteroid dehydrogenases determine their orientation in the endoplasmic reticulum membrane
The N-terminal anchor sequences of 11β-hydroxysteroid dehydrogenases determine their orientation in the endoplasmic reticulum membrane
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DOI：
10.1074/jbc.274.40.28762
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发表时间：
1999-10-01
影响因子：
4.8
通讯作者：
Frey, FJ
中科院分区：
文献类型：
--
作者：
Odermatt, A;Arnold, P;Frey, FJ
11 β-羟基类固醇脱氢酶（11 β-HSD）调节活性内源性糖皮质激素与其非活性β-代谢物的比例，从而控制糖皮质激素对其同源受体的接近。在这项研究中，通过免疫组织化学和体外转录/翻译产物的蛋白酶保护测定分析了11 β-HSD 1和11 β-HSD 2的拓扑结构和细胞内定位。用FLAG表位标记的11个β-HSD构建体在HEK-293细胞中瞬时表达。标记和天然酶的酶特性是不可区分的。荧光显微镜显示11 β-HSD 1和11 β-HSD 2仅定位于内质网（ER）膜。为了检查ER膜内标记的11 β-HSD酶的取向，我们用抗FLAG抗体对选择性透化的HEK-293细胞进行染色。免疫组化显示11 β-HSD 1的N末端位于胞浆内，含有C末端的催化结构域突出到ER腔中。相比之下，11 β-HSD 2的N末端是内腔的，并且催化结构域面向细胞质。其中11 β-HSD 1和11 β-HSD 2的N末端锚序列交换的嵌合蛋白在ER膜中采用反向取向。然而，这两种嵌合蛋白都没有催化活性。此外，在11 β-HSD 1的跨膜区段中酪氨酸基序突变为丙氨酸显著降低了V-max。11 β-HSD 1的亚细胞定位不受N端酪氨酸基序或二赖氨酸基序突变的影响。然而，残基Lys（5），而不是Lys（6），对11 β-HSD 1的拓扑结构是关键的。将Lys（5）突变为Ser，逆转了ER膜中11 β-HSD 1的取向，而不丧失催化活性。我们的研究结果强调了11 β-HSD酶的N-末端跨膜片段对于其正常功能的重要性，并表明它们足以确定它们在ER膜中的取向。
11 beta-Hydroxysteroid dehydrogenase enzymes (11 beta-HSD) regulate the ratio of active endogenous glucocorticoids to their inactive beto-metabolites, thereby controlling the access of glucocorticoids to their cognate receptors, In this study, the topology and intracellular localization of 11 beta-HSD1 and 11 beta-HSD2 have been analyzed by immunohistochemistry and protease protection assays of in vitro transcription/translation products. 11 beta-HSD constructs, tagged with the FLAG epitope, were transiently expressed in HEK-293 cells. The enzymatic characteristics of tagged and native enzymes were indistinguishable. Fluorescence microscopy demonstrated the localization of both 11 beta-HSD1 and 11 beta-HSD2 exclusively to the endoplasmic reticulum (ER) membrane. To examine the orientation of tagged 11 beta-HSD enzymes within the ER membrane, we stained selectively permeabilized HEK-293 cells with anti-FLAG antibody. Immunohistochemistry revealed that the N terminus of 11 beta-HSD1 is cytoplasmic, and the catalytic domain containing the C terminus is protruding into the ER lumen. In contrast, the N terminus of 11 beta-HSD2 is lumenal, and the catalytic domain is facing the cytoplasm, Chimeric proteins where the N-terminal anchor sequences of 11 beta-HSD1 and 11 beta-HSD2 were exchanged adopted inverted orientation in the ER membrane. However, both chimeric proteins were not catalytically active. Furthermore, mutation of a tyrosine motif to alanine in the transmembrane segment of 11 beta-HSD1 significantly reduced V-max. The subcellular localization of 11 beta-HSD1 was not affected by mutations of the tyrosine motif or of a di-lysine motif in the N terminus. However, residue Lys(5), but not Lys(6), turned out to be critical for the topology of 11 beta-HSD1. Mutation of Lys(5) to Ser inverted the orientation of 11 beta-HSD1 in the ER membrane without loss of catalytic activity. Our results emphasize the importance of the N-terminal transmembrane segments of 11 beta-HSD enzymes for their proper function and demonstrate that they are sufficient to determine their orientation in the ER membrane.