Lipid, detergent, and Coomassie Blue G-250 affect the migration of small membrane proteins in blue native gels: mitochondrial carriers migrate as monomers not dimers.
Lipid, detergent, and Coomassie Blue G-250 affect the migration of small membrane proteins in blue native gels: mitochondrial carriers migrate as monomers not dimers.
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脂质、去污剂和考马斯蓝 G-250 影响蓝色天然凝胶中小膜蛋白的迁移：线粒体载体以单体而不是二聚体的形式迁移。
DOI：
10.1074/jbc.m113.484329
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发表时间：
2013-07-26
期刊：
影响因子：
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通讯作者：
Kunji ER
中科院分区：
文献类型：
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作者：
Crichton PG;Harding M;Ruprecht JJ;Lee Y;Kunji ER
背景：线粒体载体在蓝色凝胶中的迁移被认为是二聚体。结果：在蓝色天然凝胶中观察到的高分子量物质由蛋白单体、去污剂、脂质和考马斯染色组成。结论：线粒体载体是单体而非二聚体。显著性：蓝色天然凝胶中小膜蛋白的表观质量需要显著校正。蓝色天然凝胶电泳是一种常用的方法，用于确定膜蛋白的寡聚状态。使用这种技术的研究报告说，线粒体载体是二聚体（由两个约32 kDa的单体组成），在某些情况下，可以与其他蛋白质形成生理相关的协会。在这里，我们仔细研究了酵母线粒体ADP/ATP载体AAC 3在蓝色天然凝胶中的行为。我们发现，AAC 3的表观质量的变化在洗涤剂和脂质依赖性的方式（从1060至10130 kDa），这是不相关的蛋白质的寡聚状态的变化，但反映了相关的洗涤剂-脂质胶束和考马斯亮蓝G-250在这项技术中使用的差异。仅在不太有利的增溶条件下观察到较高的低聚状态物质，与蛋白质的聚集一致。用人工共价AAC 3二聚体校准表明，观察到的溶解的AAC 3和其他线粒体载体的质量对应于单体。在蓝色天然凝胶样条件下，十二烷基麦芽糖苷中纯化的AAC 3的尺寸排阻色谱显示单体的质量为约120 kDa，但由于结合的带负电荷的染料的异常高的量而在凝胶上显得更小（约60 kDa），这增加了蛋白质-洗涤剂-染料胶束复合物的电泳迁移率。我们的研究结果表明，结合脂质，洗涤剂，考马斯染色改变凝胶上的线粒体载体的行为，这可能是真实的其他小的膜蛋白，其中相关的脂质-洗涤剂胶束是大的蛋白质的质量相比。
Background: Mitochondrial carriers were thought to be dimeric based on their migration in blue native gels. Results: The high molecular mass species observed in blue native gels are composed of protein monomers, detergent, lipid, and Coomassie stain. Conclusion: The mitochondrial carriers are monomeric not dimeric. Significance: The apparent mass of small membrane proteins in blue native gels requires significant correction. Blue native gel electrophoresis is a popular method for the determination of the oligomeric state of membrane proteins. Studies using this technique have reported that mitochondrial carriers are dimeric (composed of two ∼32-kDa monomers) and, in some cases, can form physiologically relevant associations with other proteins. Here, we have scrutinized the behavior of the yeast mitochondrial ADP/ATP carrier AAC3 in blue native gels. We find that the apparent mass of AAC3 varies in a detergent- and lipid-dependent manner (from ∼60 to ∼130 kDa) that is not related to changes in the oligomeric state of the protein, but reflects differences in the associated detergent-lipid micelle and Coomassie Blue G-250 used in this technique. Higher oligomeric state species are only observed under less favorable solubilization conditions, consistent with aggregation of the protein. Calibration with an artificial covalent AAC3 dimer indicates that the mass observed for solubilized AAC3 and other mitochondrial carriers corresponds to a monomer. Size exclusion chromatography of purified AAC3 in dodecyl maltoside under blue native gel-like conditions shows that the mass of the monomer is ∼120 kDa, but appears smaller on gels (∼60 kDa) due to the unusually high amount of bound negatively charged dye, which increases the electrophoretic mobility of the protein-detergent-dye micelle complex. Our results show that bound lipid, detergent, and Coomassie stain alter the behavior of mitochondrial carriers on gels, which is likely to be true for other small membrane proteins where the associated lipid-detergent micelle is large when compared with the mass of the protein.