Localization of 13 one-helix integral membrane proteins in photosystem II subcomplexes
Localization of 13 one-helix integral membrane proteins in photosystem II subcomplexes
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DOI：
10.1016/j.ab.2008.08.038
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发表时间：
2008-12-15
影响因子：
2.9
通讯作者：
Eichacker, Lutz Andreas
中科院分区：
文献类型：
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作者：
Granvogl, Bernhard;Zoryan, Mikael;Eichacker, Lutz Andreas
光系统 II 是叶绿体中类囊体膜的多聚体蛋白质复合物。至少26种不同的整合膜蛋白亚基中大约一半的分子量低于10kDa。经过一维 (1D) 或二维 (2D) 聚丙烯酰胺凝胶电泳 (PACE) 分离，然后对检测到的蛋白质进行酶消化后，几乎无法检测到任何这些低分子量 (LMW) 亚基。因此，我们开发了一种分析高疏水性LMW蛋白的方法。使用甲酸和有机溶剂的混合物从丙烯酰胺凝胶中提取完整的蛋白质，用丙酮沉淀，并通过“自上而下”质谱法 (MS) 进行分析。离线nanoESI（电喷雾电离）MS 后，检测到来自光系统 II 的所有 LMW 单螺旋蛋白。在烟草野生型植物的 4 个检测到的光系统 II 超复合物中，鉴定出 11 种不同的单螺旋蛋白：PsbE、-F、-H、-I、-K、-L、-M、-Tc、-W 和 PsbX 的两种亚型。蓝色天然 (BN) PAGE 后，蛋白质 PsbJ、-Y1 和 -Y2 定位于缓冲液前沿。表明它们在溶解过程中的释放。组装的 PsbW 仅在超级复合物中检测到，而在光系统 II 核心复合物中不存在，这证实了该蛋白质在光捕获复合物组装中的功能。该方法将证实凝胶印迹免疫分析可定位和鉴定任何膜蛋白复合物中的 LMW 蛋白亚基。 (C) 2008 Elsevier Inc. 保留所有权利。
Photosystem II is a multimeric protein complex of the thylakoid membrane in chloroplasts. Approximately half of the at least 26 different integral membrane protein subunits have molecular masses lower than 10 kDa. After one-dimensional (1D) or two-dimensional (2D) polyacrylamide gel electrophoresis (PACE) separation, followed by enzymatic digestion of detected proteins, hardly any of these low-molecular-weight (LMW) Subunits are detectable. Therefore, we developed a method for the analysis of highly hydrophobic LMW proteins. Intact proteins are extracted from acrylamide gels using a mixture of formic acid and organic solvent, precipitated with acetone, and analyzed by "top-down" mass spectrometry (MS). After offline nanoESI (electrospray ionization) MS, all LMW one-helix proteins from photosystem II were detected. In the four detected photosystem II supercomplexes of Nicotiana tabacum wild-type plants, 11 different one-helix proteins were identified as PsbE, -F, -H, -I, -K, -L, -M, -Tc, -W, and two isoforms of PsbX. The proteins PsbJ, -Y1, and -Y2 were localized in the buffer front after blue native (BN) PAGE. indicating their release during solubilization. Assembled PsbW is detected exclusively in supercomplexes, whereas it is absent in photosystem II core complexes, corroborating the protein's function for assembly of the light-harvesting complexes. This approach will substantiate gel-blot immunoanalysis for localization and identification of LMW protein subunits in any membrane protein complex. (C) 2008 Elsevier Inc. All rights reserved.