Department of Biology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599-3280, USA.
J Biol Chem. 2012 Oct 5;287(41):33945-55. doi: 10.1074/jbc.M112.402941. Epub 2012 Aug 24.
Vitamin K epoxide reductase (VKOR) is essential for the production of reduced vitamin K that is required for modification of vitamin K-dependent proteins. Three- and four-transmembrane domain (TMD) topology models have been proposed for VKOR. They are based on in vitro glycosylation mapping of the human enzyme and the crystal structure of a bacterial (Synechococcus) homologue, respectively. These two models place the functionally disputed conserved loop cysteines, Cys-43 and Cys-51, on different sides of the endoplasmic reticulum (ER) membrane. In this study, we fused green fluorescent protein to the N or C terminus of human VKOR, expressed these fusions in HEK293 cells, and examined their topologies by fluorescence protease protection assays. Our results show that the N terminus of VKOR resides in the ER lumen, whereas its C terminus is in the cytoplasm. Selective modification of cysteines by polyethylene glycol maleimide confirms the cytoplasmic location of the conserved loop cysteines. Both results support a three-TMD model of VKOR. Interestingly, human VKOR can be changed to a four-TMD molecule by mutating the charged residues flanking the first TMD. Cell-based activity assays show that this four-TMD molecule is fully active. Furthermore, the conserved loop cysteines, which are essential for intramolecular electron transfer in the bacterial VKOR homologue, are not required for human VKOR whether they are located in the cytoplasm (three-TMD molecule) or the ER lumen (four-TMD molecule). Our results confirm that human VKOR is a three-TMD protein. Moreover, the conserved loop cysteines apparently play different roles in human VKOR and in its bacterial homologues.
维生素 K 环氧化物还原酶（VKOR）对于还原型维生素 K 的生成至关重要，而还原型维生素 K 是维生素 K 依赖性蛋白修饰所必需的。已经提出了 VKOR 的三跨膜结构域（TMD）和四跨膜结构域拓扑模型。它们分别基于人酶的体外糖基化作图和细菌（蓝藻）同源物的晶体结构。这两种模型将功能上有争议的保守环半胱氨酸，Cys-43 和 Cys-51，放置在内质网（ER）膜的不同侧。在这项研究中，我们将绿色荧光蛋白融合到人 VKOR 的 N 或 C 末端，在 HEK293 细胞中表达这些融合蛋白，并通过荧光蛋白酶保护测定法检查它们的拓扑结构。我们的结果表明，VKOR 的 N 末端位于内质网腔中，而其 C 末端位于细胞质中。聚乙二醇马来酰亚胺对半胱氨酸的选择性修饰证实了保守环半胱氨酸的细胞质定位。这两个结果都支持 VKOR 的三 TMD 模型。有趣的是，通过突变第一个 TMD 周围的带电残基，人 VKOR 可以转变为四跨膜分子。基于细胞的活性测定表明，这种四跨膜分子是完全活跃的。此外，在细菌 VKOR 同源物中对于分子内电子转移至关重要的保守环半胱氨酸，无论是位于细胞质中（三 TMD 分子）还是内质网腔中（四 TMD 分子），对于人 VKOR 都是不需要的。我们的结果证实人 VKOR 是一种三跨膜蛋白。此外，保守环半胱氨酸在人 VKOR 及其细菌同源物中显然发挥不同的作用。
