11的氨基端锚序列beta-hydroxysteroid脱氢酶决定他们的方向内质网的膜。
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Odermatt导演,阿诺德P, Stauffer弗雷BM,弗雷陆地
11的氨基端锚序列beta-hydroxysteroid脱氢酶决定他们的方向内质网的膜。
生物化学杂志。1999年10月1日,274 (40):28762 - 70。
- PubMed ID
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10497248 (在PubMed]
- 文摘
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11 beta-hydroxysteroid脱氢酶酶(11β- HSD)的比例调节活跃不活跃的keto-metabolites内源性糖皮质激素,从而控制糖皮质激素的访问他们的同源受体。在这项研究中,拓扑结构和细胞内定位11 beta-hsd1和11 beta-hsd2被免疫组织化学分析和蛋白酶保护体外转录/翻译产品的化验。11 beta-hsd构造、标记与标记抗原决定基,是暂时性的hek - 293细胞中表达。标记和原生酶的酶的特点是没有区别的。荧光显微法证明了本地化的11 beta-hsd1和11 beta-hsd2专门的内质网(ER)膜。检查标记的方向11 beta-hsd ER内酶膜,我们染色有选择地permeabilized hek - 293细胞与anti-FLAG抗体。免疫组织化学显示,N末端的11 beta-hsd1细胞质,和催化域包含C末端突出到ER腔。相比之下,N末端11 beta-hsd2食管腔,催化领域正面临细胞质。嵌合蛋白质的氨基端锚序列11 beta-hsd1和11 beta-hsd2交换采用反向ER膜的取向。然而,嵌合蛋白质没有催化地活跃。 Furthermore, mutation of a tyrosine motif to alanine in the transmembrane segment of 11beta-HSD1 significantly reduced V(max). The subcellular localization of 11beta-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 11beta-HSD1. Mutation of Lys(5) to Ser inverted the orientation of 11beta-HSD1 in the ER membrane without loss of catalytic activity. Our results emphasize the importance of the N-terminal transmembrane segments of 11beta-HSD enzymes for their proper function and demonstrate that they are sufficient to determine their orientation in the ER membrane.