Stenberg Filippa, Chovanec Peter, Maslen Sarah L, Robinson Carol V, Ilag Leopold L, von Heijne Gunnar, Daley Daniel O
Department of Biochemistry and Stockholm University, Stockholm SE-106 91, Sweden.
J Biol Chem. 2005 Oct 14;280(41):34409-19. doi: 10.1074/jbc.M506479200. Epub 2005 Aug 3.
Protein complexes are an intrinsic aspect of life in the membrane. Knowing which proteins are assembled in these complexes is therefore essential to understanding protein function(s). Unfortunately, recent high throughput protein interaction studies have failed to deliver any significant information on proteins embedded in the membrane, and many membrane protein complexes remain ill defined. In this study, we have optimized the blue native-PAGE technique for the study of membrane protein complexes in the inner and outer membranes of Escherichia coli. In combination with second dimension SDS-PAGE and mass spectrometry, we have been able to identify 43 distinct protein complexes. In addition to a number of well characterized complexes, we have identified known and orphan proteins in novel oligomeric states. For two orphan proteins, YhcB and YjdB, our findings enable a tentative functional assignment. We propose that YhcB is a hitherto unidentified additional subunit of the cytochrome bd oxidase and that YjdB, which co-localizes with the ZipA protein, is involved in cell division. Our reference two-dimensional blue native-SDS-polyacrylamide gels will facilitate future studies of the assembly and composition of E. coli membrane protein complexes during different growth conditions and in different mutant backgrounds.
蛋白质复合物是膜生命活动的一个内在方面。因此，了解哪些蛋白质组装在这些复合物中对于理解蛋白质功能至关重要。不幸的是，最近的高通量蛋白质相互作用研究未能提供关于嵌入膜中的蛋白质的任何重要信息，许多膜蛋白复合物仍然定义不明确。在本研究中，我们优化了蓝色非变性聚丙烯酰胺凝胶电泳技术，用于研究大肠杆菌内膜和外膜中的膜蛋白复合物。结合二维十二烷基硫酸钠聚丙烯酰胺凝胶电泳和质谱分析，我们已经能够鉴定出43种不同的蛋白质复合物。除了一些特征明确的复合物外，我们还鉴定出处于新型寡聚状态的已知和孤儿蛋白。对于两种孤儿蛋白YhcB和YjdB，我们的发现使我们能够进行初步的功能分配。我们提出，YhcB是细胞色素bd氧化酶迄今未被识别的额外亚基，并且与ZipA蛋白共定位的YjdB参与细胞分裂。我们的参考二维蓝色非变性-十二烷基硫酸钠聚丙烯酰胺凝胶将有助于未来对不同生长条件和不同突变背景下大肠杆菌膜蛋白复合物的组装和组成的研究。
