Membrane proteomics of phagosomes suggests a connection to autophagy

Abstract

Phagocytosis is the central process by which macrophage cells internalize and eliminate infectious microbes as well as apoptotic cells. During maturation, phagosomes containing engulfed particles fuse with various endosomal compartments through the action of regulatory molecules on the phagosomal membrane. In this study, we performed a proteomic analysis of the membrane fraction from latex bead-containing (LBC) phagosomes isolated from macrophages. The profile, which comprised 546 proteins, suggests diverse functions of the phagosome and potential connections to secretory processes, toll-like receptor signaling, and autophagy. Many identified proteins were not previously known to reside in the phagosome. We characterized several proteins in LBC phagosomes that change in abundance on induction of autophagy, a process that has been previously implicated in the host defense against microbial pathogens. These observations suggest crosstalk between autophagy and phagocytosis that may be relevant to the innate immune response of macrophages.LC3 ͉ phagocytosis P hagosomes are specialized membrane-bound organelles generated in phagocytic cells such as macrophages, neutrophils, and dendritic cells. Their purpose is to internalize foreign particles, microorganisms, or apoptotic cells, to mount an immune response, or to maintain tissue homeostasis (1). The nascent phagosome undergoes a complex maturation process involving sequential fusion with endosomal compartments. Once mature, the phagosome degrades its constituents and facilitates antigen presentation in a highly controlled manner (2-4). Insight into the biogenesis and immunity-related functions of the phagosome has come from analysis of its protein contents. Previous studies have profiled the proteomes of latex bead-containing (LBC) phagosomes in cell lines from mice (5-7) and Drosophila sp. (8), as well as Dictyostelium discoideum (9) and Entamoeba histolytica (10). These elegant studies contribute to our understanding of phagosome maturation (6, 9) and modulation by cytokines (7); however, because the entire contents of the phagosomes were sampled, abundant proteins, such as soluble lysosomal hydrolases, might have obscured lower abundance membrane-bound regulatory proteins or signaling factors.To identify such lower abundance species, we enriched integral and peripheral membrane proteins from macrophage LBC phagosomes by organelle sub-fractionation, before carrying out a large-scale proteomic analysis. The 546 proteins identified in our study included 49 membrane receptors, 64 transporter proteins, 107 regulators of vesicle and protein trafficking (including GTPases), and many components from cellular machineries other than phagosomes. One of the new proteins exclusively found in our study, LC3-II, is considered a marker of autophagic activity. Its presence in phagosomes suggests an unexplored linkage between autophagy and phagocytosis. Apart from its housekeeping role in maintaining cellular homeostasis, autophagy has been implicated in cancer, neurodegerative di...

The Role of CD38 in Fcγ Receptor (FcγR)-mediated Phagocytosis in Murine Macrophages

“…After cell disruption using 30-gauge needle syringes, the latex bead containing phagosomes were isolated on a sucrose gradient (52,53). The isolated phagosome was washed and resuspended with serum-free media for further use in experiments.…”

Proteomic Characterization of Phagosomal Membrane Microdomains During Phagolysosome Biogenesis and Evolution

“…We have shown previously that phagosome isolated by flotation display low levels of contamination (40). The recent finding that phagosomes extensively interact with autophagosomes (41,42) suggests that this type of interaction would bring a wide variety of proteins to the phagosome lumen for degradation. Interestingly, we observed that phagosomes isolated from cells displaying high levels of autophagic activity contained higher levels of the aforementioned types of proteins (unpublished observations).…”

Autophagy as a macrophage response to bacterial infection

“…Proteomic analysis of isolated phagosomal membranes from naïve uninfected macrophages revealed the presence of LC3, the level of which increases in response to starvation-induced autophagy. This result suggests a direct relationship between phagocytosis and autophagy, and possibly a more general role for LAP in the physiology of macrophages (74). Indeed, LAP plays an important role in the autoimmune response by efficient phagocytic clearance of apoptotic cells (72).…”