Say Yee-How, Hooper Nigel M
Proteolysis Research Group, Institute of Molecular and Cellular Biology, Faculty of Biological Sciences, University of Leeds, Leeds, UK.
Proteomics. 2007 Apr;7(7):1059-64. doi: 10.1002/pmic.200600849.
Subcellular fractionation is central to a range of cell biological, biochemical and proteomic studies. Purification of nuclear-enriched fractions is critical for studies on nuclear structure and function. Here we show that detergent-based nuclear isolation methods cause the redistribution of proteins associated with plasma membrane lipid rafts into nuclear fractions. The glycosyl-phosphatidylinositol (GPI)-anchored prion protein (PrP(C)) and a GPI-anchored construct of angiotensin converting enzyme (GPI-ACE), as well as the lipid raft markers flotillin-1 and -2, were present in the nuclear fractions derived using three different subcellular fractionation protocols. Incubation of intact cells with bacterial phosphatidylinositol-specific phospholipase C (PI-PLC), which cleaves GPI-anchored proteins from the cell surface, significantly reduced the amount of PrP(C) and GPI-ACE in the nuclear fraction. Buoyant sucrose density gradient centrifugation in the presence of Triton X-100 of the nuclear fraction resulted in a significant proportion of the GPI-anchored proteins being recovered in the low density lipid raft fractions. These data indicate that the nuclear fraction isolated using such subcellular fractionation protocols is contaminated with components of plasma membrane lipid rafts and raises questions as to the integrity of the nuclear fraction isolated by such protocols for use in detailed cell biological studies and proteomics analysis.
亚细胞分级分离对于一系列细胞生物学、生物化学和蛋白质组学研究至关重要。富含细胞核的组分的纯化对于细胞核结构与功能的研究至关重要。在此我们表明，基于去污剂的细胞核分离方法会导致与质膜脂筏相关的蛋白质重新分布到细胞核组分中。糖基磷脂酰肌醇（GPI）锚定的朊病毒蛋白（PrP(C)）和血管紧张素转换酶的GPI锚定构建体（GPI-ACE），以及脂筏标记物flotillin-1和flotillin-2，存在于使用三种不同亚细胞分级分离方案获得的细胞核组分中。用细菌磷脂酰肌醇特异性磷脂酶C（PI-PLC）处理完整细胞，该酶可从细胞表面切割GPI锚定蛋白，显著降低了细胞核组分中PrP(C)和GPI-ACE的量。在Triton X-100存在下对细胞核组分进行浮力蔗糖密度梯度离心，导致相当一部分GPI锚定蛋白在低密度脂筏组分中被回收。这些数据表明，使用此类亚细胞分级分离方案分离的细胞核组分被质膜脂筏的成分污染，这对使用此类方案分离的细胞核组分在详细的细胞生物学研究和蛋白质组学分析中的完整性提出了疑问。
