Analysis of membrane-protein complexes of the marine sulfate reducer Desulfobacula toluolica Tol2 by 1D blue native-PAGE complexome profiling and 2D blue native-/SDS-PAGE
Analysis of membrane-protein complexes of the marine sulfate reducer Desulfobacula toluolica Tol2 by 1D blue native-PAGE complexome profiling and 2D blue native-/SDS-PAGE
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DOI：
10.1002/pmic.201500360
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发表时间：
2016-03-01
期刊：
影响因子：
3.4
通讯作者：
Rabus, Ralf
中科院分区：
文献类型：
--
作者：
Woehlbrand, Lars;Ruppersberg, Hanna S.;Rabus, Ralf
硫酸盐还原菌（SRB）通过APS还原酶（AprAB）和异化亚硫酸盐还原酶（DsrAB）将硫酸盐还原为硫化物来获得能量。这些酶被预测从膜氧化还原复合物，即醌相互作用的膜结合氧化还原酶（QmoABC）和DsrMKJOP复合物获得电子。除了这些保守的复合物，SRB的基因组编码大量的其他（预测）膜氧化还原复合物，其功能和实际形成是未知的。建立了海洋硫酸盐还原菌甲苯脱硫杆菌（Desulfobacula toluolica）Tol 2膜蛋白复合体的1D Blue Native-PAGE和2D BN-/SDS-PAGE分析方法。对>800种蛋白质的标准化评分谱的分析结合分级聚类和2D BN-/SDS-PAGE分离点的鉴定证明了膜复合物以其天然形式（例如ATP合酶）的分离。除了QmoABC和DsrMKJOP复合物外，还检测到了构成D. toluolica Tol 2，例如转运蛋白（例如钠/硫酸盐共转运蛋白）或涉及基于Na+的生物能量学的氧化还原复合物（RnfABCDEG）。值得注意的是，尺寸估计表明DsrMKJOP复合物在体内形成二聚体和四聚体。此外，聚类分析表明，该复合物的相互作用与硫氰酸酯样蛋白（Tol2_C05230）可能代表周质电子传递合作伙伴DsrMKJOP。
Sulfate-reducing bacteria (SRB) obtain energy from cytoplasmic reduction of sulfate to sulfide involving APS-reductase (AprAB) and dissimilatory sulfite reductase (DsrAB). These enzymes are predicted to obtain electrons from membrane redox complexes, i.e. the quinone-interacting membrane-bound oxidoreductase (QmoABC) and DsrMKJOP complexes. In addition to these conserved complexes, the genomes of SRB encode a large number of other (predicted) membrane redox complexes, the function and actual formation of which is unknown. This study reports the establishment of 1D Blue Native-PAGE complexome profiling and 2D BN-/SDS-PAGE for analysis of the membrane protein complexome of the marine sulfate reducer Desulfobacula toluolica Tol2. Analysis of normalized score profiles of >800 proteins in combination with hierarchical clustering and identification of 2D BN-/SDS-PAGE separated spots demonstrated separation of membrane complexes in their native form, e.g. ATP synthase. In addition to the QmoABC and DsrMKJOP complexes, other complexes were detected that constitute the basic membrane complexome of D. toluolica Tol2, e.g. transport proteins (e.g. sodium/sulfate symporters) or redox complexes involved in Na+-based bioenergetics (RnfABCDEG). Notably, size estimation indicates dimer and quadruple formation of the DsrMKJOP complex in vivo. Furthermore, cluster analysis suggests interaction of this complex with a rhodanese-like protein (Tol2_C05230) possibly representing a periplasmic electron transfer partner for DsrMKJOP.