Topology of prostaglandin H synthase-1 in the endoplasmic reticulum membrane.
Topology of prostaglandin H synthase-1 in the endoplasmic reticulum membrane.
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内质网膜中前列腺素 H 合酶 1 的拓扑结构。
DOI：
10.1006/abbi.1995.0027
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发表时间：
1995
期刊：
影响因子：
--
通讯作者：
Kulmacz,RJ
中科院分区：
文献类型：
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作者：
Ren,Y;Walker,C;Loose-Mitchell,DS;Deng,J;Ruan,KH;Kulmacz,RJ
前列腺素H合酶-1是一种整合的内质网膜蛋白，其催化前列腺素生物合成中的关键控制步骤。前列腺素H合酶-1多肽相对于内质网膜的整体安排进行了检查，在瞬时转染COS-1细胞，使用免疫荧光显微镜。细菌毒素，链球菌溶血素-O，用于选择性质膜透化和洗涤剂，皂苷，一般膜透化。用特异于特定前列腺素H合酶-1肽段的六种抗体和特异于插入的病毒报告表位的一种抗体探测处理的细胞。对照实验确定，肌动蛋白，细胞质标记物，是可访问的荧光素标记鬼笔环肽后，链球菌溶血素-O治疗，而抗体对蛋白质二硫键异构酶，内质网内腔标记物，结合后，只有皂苷治疗。使用这种方法来研究前列腺素H合酶-1，它被发现，链球菌溶血素-O的治疗是足够的，以获得细胞内膜的内源性C-末端片段的特异性抗体染色，插入在C-末端的病毒报告，和arg 277附近的蛋白酶敏感区域。相反，皂苷处理对于通过对跨越残基51-66、156-170和377-390的肽具有特异性的抗体染色是必要的。靶向残基483-496的抗体即使在皂苷透化后也不染色转染的细胞，尽管它们确实与洗涤剂溶解的前列腺素H合酶-1结合。这些结果表明，C-末端和arg 277区域的合酶可以暴露在内质网膜的细胞质侧，而区域附近的N-糖基化位点被限制在内质网腔和残基483-496是从内质网膜的两侧无法访问。
Prostaglandin H synthase-1 is an integral endoplasmic reticulum membrane protein which catalyzes a key control step in prostaglandin biosynthesis. The overall arrangement of the prostaglandin H synthase-1 polypeptide with respect to the endoplasmic reticulum membrane was examined in transiently transfected COS-1 cells, using immunofluorescence microscopy. A bacterial toxin, streptolysin-O, was used for selective plasma membrane permeabilization and a detergent, saponin, for general membrane permeabilization. Treated cells were probed with six antibodies specific for particular prostaglandin H synthase-1 peptide segments and one antibody specific for an inserted viral reporter epitope. Control experiments established that actin, a cytoplasmic marker, was accessible to fluorescein-labeled phalloidin after streptolysin-O treatment, whereas antibodies against protein disulfide isomerase, an endoplasmic reticulum lumenal marker, bound only after saponin treatment. Using this approach to investigate prostaglandin H synthase-1, it was found that streptolysin-O treatment was sufficient to obtain staining of intracellular membranes by antibodies specific for the endogenous C-terminal segment, for the viral reporter inserted at the C-terminus, and for the protease-sensitive region near arg277. In contrast, saponin treatment was necessary for staining by antibodies specific for peptides spanning residues 51–66, 156–170, and 377–390. Antibodies targeted against residues 483–496 did not stain transfected cells even after saponin permeabilization, although they did bind to detergent-solubilized prostaglandin H synthase-1. These results indicate that the C-terminus and arg277 regions of the synthase can be exposed on the cytoplasmic side of the endoplasmic reticulum membrane, whereas regions near N-glycosylation sites are confined to the endoplasmic reticulum lumen and residues 483–496 are inaccessible from either side of the endoplasmic reticulum membrane.