Human ABCB6 localizes to both the outer mitochondrial membrane and the plasma membrane
Human ABCB6 localizes to both the outer mitochondrial membrane and the plasma membrane
复制标题
DOI：
10.1021/bi700015m
复制
发表时间：
2007-08-21
期刊：
影响因子：
2.9
通讯作者：
Gottesman, Michael M.
中科院分区：
文献类型：
--
作者：
Paterson, Jill K.;Shukla, Suneet;Gottesman, Michael M.
ATP结合盒转运蛋白ABCB 6的表达与多种细胞功能相关，包括对多种细胞毒性药物的耐药性、铁稳态和卟啉转运。为了进一步阐明其生理功能和/或在耐药性中的作用，我们确定了ABCB 6的亚细胞定位。使用三种新的ABCB6特异性抗体，表达cDNA衍生的或内源性ABCB6的细胞的Western印迹分析揭示了两种不同的分子量形式。共聚焦显微镜表明，蛋白质定位于线粒体和质膜。差速离心显示，低分子量形式主要存在于线粒体中，而较大的蛋白质形式在质膜中更丰富。初步研究表明，ABCB 6在质膜中具有功能相关性，其表达可防止细胞中特定卟啉的积累。毛地黄皂苷溶解的线粒体表明，ABCB6是存在于外线粒体膜，而与ABCB6特异性抗体的反滴定分析表明，ABCB6的核苷酸结合结构域是细胞质。这些研究首次证明ABCB 6以两种分子量形式存在，定位于线粒体外膜和质膜，并在质膜中发挥功能作用。
Expression of the ATP-binding cassette transporter ABCB6 has been associated with multiple cellular functions, including resistance to several cytotoxic agents, iron homeostasis, and porphyrin transport. To further elucidate its physiological function and/or role in drug resistance, we determined the subcellular location of ABCB6. Using three novel ABCB6-specific antibodies, Western blot analysis of cells expressing cDNA-derived or endogenous ABCB6 revealed two distinct molecular weight forms. Confocal microscopy indicates that the protein localizes to both mitochondria and the plasma membrane. Differential centrifugation revealed that the lower molecular weight form predominantly resides in the mitochondria, while the larger protein form is more abundant in the plasma membrane. Preliminary studies indicate that ABCB6 is functionally relevant in the plasma membrane, where its expression prevents the accumulation of specific porphyrins in the cell. Digitonin solubilization of mitochondria demonstrated that ABCB6 is present in the outer mitochondrial membrane, while back-titration assays with the ABCB6-specific antibodies reveal that the nucleotide binding domain of ABCB6 is cytoplasmic. These studies are the first to demonstrate that ABCB6 exists in two molecular weight forms, is localized to both the outer mitochondrial membrane and the plasma membrane, and plays a functional role in the plasma membrane.