Candida drug resistance protein 1, a major multidrug ATP binding cassette transporter of Candida albicans, translocates fluorescent phospholipids in a reconstituted system.
Candida drug resistance protein 1, a major multidrug ATP binding cassette transporter of Candida albicans, translocates fluorescent phospholipids in a reconstituted system.
复制标题
DOI：
10.1021/bi700453e
复制
发表时间：
2007-10
期刊：
影响因子：
2.9
通讯作者：
S. Shukla;V. Rai;P. Saini;D. Banerjee;A. Menon;R. Prasad
中科院分区：
文献类型：
--
作者：
S. Shukla;V. Rai;P. Saini;D. Banerjee;A. Menon;R. Prasad
白色念珠菌耐药蛋白 1 (Cdr1p) 是一种 ATP 依赖性药物外排泵，有助于念珠菌感染的免疫功能低下患者的多重耐药性。先前基于细胞的检测表明 Cdr1p 也可充当磷脂转运蛋白。为了研究这一点，我们将纯化的 Cdr1p 重组到密封的膜囊泡中。密封和透化蛋白脂质体的 ATP 酶活性比较表明，Cdr1p 是不对称重构的，因此大约 70% 的分子具有可进入囊外空间的 ATP 结合位点。荧光甘油磷脂被掺入脂蛋白体的外叶中，并使用不透膜的连二亚硫酸盐通过淬灭测定来追踪它们向内叶的转运。我们观察到荧光脂质依赖 ATP 转运至囊泡内层。每个囊泡平均约有 6 个 Cdr1p 分子，达到最大转运程度的半衰期约为 15 分钟。在用 ATP 酶活性受损的 Cdr1p 变体重建的囊泡中，运输减少，并且可以通过摩尔过量的药物（例如已知会被 Cdr1p 流出的氟康唑和咪康唑）竞争到不同水平。转运不受氨苄青霉素的影响，氨苄青霉素是一种不被 Cdr1p 外排的化合物。我们的结果表明 Cdr1p 转运荧光磷脂的能力与外排药物之间存在直接联系。我们注意到，念珠菌 ABC 超家族中只有少数成员具有明确的药物出口者角色；因此，Cdr1p介导的脂质易位可以反映其细胞功能。
Candida albicans drug resistance protein 1 (Cdr1p), an ATP-dependent drug efflux pump, contributes to multidrug resistance in Candida-infected immunocompromised patients. Previous cell-based assays suggested that Cdr1p also acts as a phospholipid translocator. To investigate this, we reconstituted purified Cdr1p into sealed membrane vesicles. Comparison of the ATPase activities of sealed and permeabilized proteoliposomes indicated that Cdr1p was asymmetrically reconstituted such that approximately 70% of the molecules had their ATP binding sites accessible to the extravesicular space. Fluorescent glycerophospholipids were incorporated into the outer leaflet of the proteoliposomes, and their transport into the inner leaflet was tracked with a quenching assay using membrane-impermeant dithionite. We observed ATP-dependent transport of the fluorescent lipids into the inner leaflet of the vesicles. With approximately 6 molecules of Cdr1p per vesicle on average, the half-time to reach the maximal extent of transport was approximately 15 min. Transport was reduced in vesicles reconstituted with Cdr1p variants with impaired ATPase activity and could be competed out to different levels by a molar excess of drugs such as fluconazole and miconazole that are known to be effluxed by Cdr1p. Transport was not affected by ampicillin, a compound that is not effluxed by Cdr1p. Our results suggest a direct link between the ability of Cdr1p to translocate fluorescent phospholipids and efflux drugs. We note that only a few members of the ABC superfamily of Candida have a well-defined role as drug exporters; thus, lipid translocation mediated by Cdr1p could reflect its cellular function.