The endoplasmic reticulum (ER) is thought to play an important structural and functional role in phagocytosis. According to this model, direct membrane fusion between the ER and the plasma or phagosomal membrane must precede further invagination, but the exact mechanisms remain elusive. Here, we investigated whether various ER-localized SNARE proteins are involved in this fusion process. When phagosomes were isolated from murine J774 macrophages, we found that ER-localized SNARE proteins (syntaxin 18, D12, and Sec22b) were significantly enriched in the phagosomes. Fluorescence and immuno-EM analyses confirmed the localization of syntaxin 18 in the phagosomal membranes of J774 cells stably expressing this protein tagged to a GFP variant. To examine whether these SNARE proteins are required for phagocytosis, we generated 293T cells stably expressing the Fc␥ receptor, in which phagocytosis occurs in an IgGmediated manner. Expression in these cells of dominant-negative mutants of syntaxin 18 or D12 lacking the transmembrane domain, but not a Sec22b mutant, impaired phagocytosis. Syntaxin 18 small interfering RNA (siRNA) selectively decreased the efficiency of phagocytosis, and the rate of phagocytosis was markedly enhanced by stable overexpression of syntaxin 18 in J774 cells. Therefore, we conclude that syntaxin 18 is involved in ER-mediated phagocytosis, presumably by regulating the specific and direct fusion of the ER and plasma or phagosomal membranes. INTRODUCTIONPhagocytosis is the coordinated process by which large foreign particles are internalized into a newly formed organelle, the phagosome. In professional phagocytes of the immune system, including macrophages, neutrophils, and dendritic cells, the internalization is triggered by activation of various types of cell surface receptors in the course of innate and adaptive immune responses. For example, in the case of immunoglobulin (Ig)-mediated phagocytosis, Fc receptors (Fc␥Rs) cluster where they contact an Ig-opsonized solid surface; this induces actin polymerization, resulting in the formation of pseudopods that engulf the particle . Subsequently, the phagosomes mature by fusing with other organelles of the endocytic pathway, leading to the formation of phagolysosomes (Downey et al., 1999;Vieira et al., 2002).In macrophages, every membrane fusion event during phagocytosis is thought to be mediated by soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins (Coppolino et al., 2001). It is currently believed that the assembly of SNARE proteins leads to a tight connection between the vesicle and the target membrane, which initiates the opening of the fusion pore . The SNARE complex forms an extended parallel four-helix bundle in order to fuse the two membranes Antonin et al., 2002). Three helices are extended from one membrane by proteins of the syntaxin and SNAP-25 families (Q-SNAREs) that contain a conserved glutamine residue at a central position called the "0" layer, and the remaining helix, extended from the opposite membr...

The endoplasmic reticulum (ER) is proposed to be a membrane donor for phagosome formation. In support of this, we have previously shown that the expression level of syntaxin 18, an ER-localized SNARE protein, correlates with phagocytosis activity. To obtain further insights into the involvement of the ER in phagocytosis we focused on Sec22b, another ER-localized SNARE protein that is also found on phagosomal membranes. In marked contrast to the effects of syntaxin 18, we report here that phagocytosis was nearly abolished in J774 macrophages stably expressing mVenus-tagged Sec22b, without affecting the cell surface expression of the Fc receptor or other membrane proteins related to phagocytosis. Conversely, the capacity of the parental J774 cells for phagocytosis was increased when endogenous Sec22b expression was suppressed. Domain analyses of Sec22b revealed that the R-SNARE motif, a selective domain for forming a SNARE complex with syntaxin18 and/or D12, was responsible for the inhibition of phagocytosis. These results strongly support the ER-mediated phagocytosis model and indicate that Sec22b is a negative regulator of phagocytosis in macrophages, most likely by regulating the level of free syntaxin 18 and/or D12 at the site of phagocytosis.

The incidence of hearing loss in children due to mumps was 7/7400 (approximately 1/1000 cases), which is higher than previously suggested. Prevention of deafness is another important reason for assuring universal immunization against mumps.

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