Phagosomes, by killing and degrading pathogens for antigen presentation, are organelles implicated in key aspects of innate and adaptive immunity. While it has been well established that phagosomes consist of membranes from the plasma membrane (PM), endosomes and lysosomes (endo/lyso), 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 estimate accurately 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 post-nuclear 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 5 organelles (e.g. PM, endo/lyso, ER, Golgi and mitochondria). This data in combination with a SILAC method designed to detect potential contaminant sources revealed that the ER is part of the phagosomal membrane and contributes ~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, while that containing the derived SNARE protein mVenus-Stx18 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.
This article is published online in Molecular and Cellular Proteomics and is free to access.