The dynamic phagosomal proteome and the contribution of the endoplasmic reticulum

Abstract

Macrophages use phagocytosis to control the spread of pathogens in the body, to clear apoptotic cells, and to aid in tissue remodeling. The phagosomal membrane is traditionally thought to originate from the plasmalemma and then go through a series of maturation steps involving sequential fusion with endosomal compartments, leading to the formation of a phagolysosome. A recent model suggests that the endoplasmic reticulum (ER) is involved in the maturation as well. Here we use stable isotope labeling and multiple quantitative proteomic approaches to follow the dynamic composition of the maturing phagosome in RAW 264.7 macrophage cells to a greater depth and higher temporal resolution than was previously possible. Analysis of the results suggests that the traditional model of a linear sequence of fusion events with different compartments is more complex or variable than previously thought. By concomitantly measuring the degree to which each component is enriched on phagosomes, our data argue that the amount of ER involved in phagocytosis is much less than predicted by the model of ER-mediated phagocytosis.innate immunity ͉ organelle ͉ phagocytosis ͉ stable isotope labeling ͉ latex bead vacuoles

Size-dependent mechanism of cargo sorting during lysosome-phagosome fusion is controlled by Rab34

“…Although Rab34 has been associated to phagosome maturation (9,14), its specific function in this process has not been defined.…”

“…Multiple Rab proteins are associated with phagosomes regulating the interactions of these organelles with endosomes and lysosomes (5)(6)(7)(8). Many of these phagosomal Rab proteins are even recruited at the same time, although in some cases the kinetics is different (7,9). Although the recruitment of several Rab GTPases is reported, the function of many individual phagosome-associated Rab proteins that contribute to phagosome maturation is poorly defined.…”

“…Rab34 has also been associated with the post-Golgi secretory pathway (13) and with phagosomes (9,14). Additionally, Rab34 has been reported to interact with the vesicle priming protein mammalian uncoordinated 13-2 (Munc13-2) regulating secretion during hyperglycemia (13,15).…”

The Protein Tyrosine Phosphatase SHP-1 Regulates Phagolysosome Biogenesis

“…Indeed, characterization of the phagosome phosphoproteome and molecular analysis of the evolution of phagosomes highlighted the importance of phosphorylation in the regulation of phagolysosome biogenesis (4,12,22). This process is accomplished through transient, highly regulated interactions between the phagosome and the cytoskeleton, as well as the early, late, and recycling endocytic compartments (46)(47)(48). Through these interactions, phagosomes acidify and sequentially acquire an array of hydrolases, culminating in the generation of a hydrolytic, microbicidal environment (37,49,50).…”

Quantitative Organelle Proteomics of MCF-7 Breast Cancer Cells Reveals Multiple Subcellular Locations for Proteins in Cellular Functional Processes

“…[24][25][26][27][28] The most common approach has been purification of individual organelles followed by exhaustive determination of the protein content. The main disadvantages of this approach are that the degree of purification/ contamination of the organelle is difficult to ascertain conclusively for lower abundance proteins, that the protein content may be altered by the purification process and that the approach is not very suitable for dynamic studies of protein subcellular location.…”