Quantitative Proteomics Reveals That Only a Subset of the Endoplasmic Reticulum Contributes to the Phagosome
Quantitative Proteomics Reveals That Only a Subset of the Endoplasmic Reticulum Contributes to the Phagosome
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DOI：
10.1074/mcp.m111.016378
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发表时间：
2012-07-01
影响因子：
7
通讯作者：
Desjardins, Michel
中科院分区：
文献类型：
--
作者：
Campbell-Valois, Francois-Xavier;Trost, Matthias;Desjardins, Michel
吞噬体通过杀死和降解病原体来呈递抗原，是与先天性和适应性免疫的关键方面有关的细胞器。尽管已经明确吞噬体由质膜、内体和溶酶体的膜组成，但内质网 (ER) 膜在吞噬体的形成中发挥重要作用的观点仍存在争议。然而，缺乏准确估计潜在来源细胞器和污染物对吞噬体蛋白质组的贡献的方法。在此，我们开发了一种蛋白质组学方法，通过将这些组分与 J774A.1 鼠巨噬细胞系中的总膜和核后上清液进行比较，客观地量化各种细胞器对早期和晚期吞噬体的贡献。使用定量无标记质谱法，估计了与数百种蛋白质相对应的肽的丰度，并将其归因于五种细胞器（例如质膜、内体/溶酶体、内质网、高尔基体和线粒体）之一。这些数据与旨在检测潜在污染物源的细胞培养方法中的稳定同位素标记相结合，揭示了 ER 是吞噬体膜的一部分，并贡献了与早期吞噬体蛋白质组相似的 20%。此外，只有一部分 ER 蛋白被招募到吞噬体，这表明 ER 的特定子结构域可能参与吞噬作用。蛋白质印迹和免疫荧光充分验证了这一结论；我们能够证明，内质网标记物 GFP-KDEL 积累的内质网部分被排除在吞噬体之外，而含有 mVenus-Syntaxin 18 的内质网部分被招募。这些结果凸显了描述利什曼原虫、布鲁氏菌和军团菌致病机制的有希望的新途径，这些细菌在富含内质网的吞噬体中茁壮成长。分子与细胞蛋白质组学 11：10.1074/mcp.M111.016378，1-13，2012。
Phagosomes, by killing and degrading pathogens for antigen presentation, are organelles implicated in key aspects of innate and adaptive immunity. Although it has been well established that phagosomes consist of membranes from the plasma membrane, endosomes, and lysosomes, the notion that the endoplasmic reticulum (ER) membrane could play an important role in the formation of the phagosome is debated. However, a method to accurately estimate the contribution of potential source organelles and contaminants to the phagosome proteome has been lacking. Herein, we have developed a proteomic approach for objectively quantifying the contribution of various organelles to the early and late phagosomes by comparing these fractions to their total membrane and postnuclear supernatant of origin in the J774A.1 murine macrophage cell line. Using quantitative label-free mass spectrometry, the abundance of peptides corresponding to hundreds of proteins was estimated and attributed to one of five organelles (e. g. plasma membrane, endosomes/lysosomes, ER, Golgi, and mitochondria). These data in combination with a stable isotope labeling in cell culture method designed to detect potential contaminant sources revealed that the ER is part of the phagosomal membrane and contributes similar to 20% of the early phagosome proteome. In addition, only a subset of ER proteins is recruited to the phagosome, suggesting that a specific subdomain(s) of the ER might be involved in phagocytosis. Western blotting and immunofluorescence substantially validated this conclusion; we were able to demonstrate that the fraction of the ER in which the ER marker GFP-KDEL accumulates is excluded from the phagosomes, whereas that containing the mVenus-Syntaxin 18 is recruited. These results highlight promising new avenues for the description of the pathogenic mechanisms used by Leishmania, Brucella, and Legionella spp., which thrive in ER-rich phagosomes. Molecular & Cellular Proteomics 11: 10.1074/mcp.M111.016378, 1-13, 2012.