Involvement of a Novel Q-SNARE, D12, in Quality Control of the Endomembrane System

Okumura, Akiko; Hatsuzawa, Kiyotaka; Tamura, Taku; Nagaya, Hisao; Saeki, Kazuko; Okumura, Fumihiro; Nagao, Kazuharu; Nishikawa, Mitsuo; Yoshimura, Akihiko; Wada, Ikuo

doi:10.1074/jbc.m509715200

Cited by 27 publications

(40 citation statements)

References 58 publications

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“…Our functional analysis suggested that two peripheral syntaxin-18-binding proteins, ZW10 and RINT1, are involved in transport between the ER and Golgi complex (Arasaki et al, 2006;Arasaki et al, 2007;Hirose et al, 2004), and that BNIP1 participates in the formation of the three-way junctions of the ER network (Nakajima et al, 2004). Recent studies demonstrated that syntaxin 18 and p31 also participate in phagocytosis and post-Golgi transport (Hatsuzawa et al, 2006;Okumura et al, 2006). The versatile ability of the syntaxin 18 complex may be related to a unique mechanism of SNARE core assembly (Aoki et al, 2008).…”

Section: Introductionmentioning

confidence: 99%

Role of syntaxin 18 in the organization of endoplasmic reticulum subdomains

Iinuma

Aoki

Arasaki

et al. 2009

Journal of Cell Science

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In Syn18(390)-transfected cells, we frequently (40-50% of cells at 72 hours after transfection) observed large patches positive for an ER membrane protein, Bap31 (Annaert et al., 1997) (Fig. 1B, middle row, left). Albeit much less frequently, similar patches were observed in cells transfected with the less efficient siRNA Syn18(770) (bottom row, left), suggesting that the redistribution of Bap31 is a consequence of syntaxin 18 depletion, and not a consequence of off target effect of Syn18(390). The different frequencies of the Bap31-positive patches are probably the result of the different knockdown efficiency of the two siRNAs. Fig. 1B also shows that silencing of syntaxin 18 causes a substantial dispersion of the Golgi complex marked by a cis-Golgi marker, p115 (Waters et al., 1992), without affecting microtubules. Other Golgi proteins, such as GM130, mannosidase II (Man II), β-COP and the KDEL receptor (KDEL-R), were also dispersed (supplementary material Fig. S1). The time course of morphological changes of the ER and Golgi structures concomitant with syntaxin 18 depletion is shown in supplementary material Fig. S2.To investigate in detail the morphology of the ER and the Golgi complex in syntaxin-18-depleted cells, we performed electron microscopy. In Syn18(390)-transfected cells, vesiculated membrane structures, instead of the Golgi stacks, were observed at the perinuclear region ( Fig. 2B,C; supplementary material Fig. S3). Furthermore, there were well-defined membrane aggregates consisting of a convoluted network of branching tubules, as well as dilated ER structures, in Syn18(390)-transfected cells ( Fig. 2B-D; supplementary material Fig. S3). Similar results were obtained with Syn18(770)-transfected cells, although ER aggregates were observed only in some cells (data not shown). Quantitative analysis showed that the area and length of the ER normalized to the cytoplasmic area of Syn18(390)-transfected cells are higher than those of mock-transfected cells (Tables 1 and 2), suggesting a proliferation of the ER membrane concomitant with syntaxin 18 depletion.Immunoelectron microscopy confirmed Golgi disassembly and the formation of ER membrane aggregates in syntaxin-18-depleted cells. In Syn18(390)-transfected cells, a cis-Golgi marker, p115, and . At 72 hours after transfection, the cells were stained with an antibody against syntaxin 18 (right) or solubilized in phosphate-buffered saline with 0.5% SDS. The lysates (10 μg each) were separated by SDS-PAGE and analyzed by immunoblotting with the indicated antibodies (left). (B) HeLa cells were treated as described in A and stained for Bap31, p115 or α-tubulin. The distributions of the proteins investigated were indistinguishable between mock-transfected cells and lamin A/C siRNA-treated cells (data not shown). Scale bars: 10 μm. The boxed area in B is shown enlarged in C. G, Golgi complex; M, mitochondria; ER, endoplasmic reticulum; N, nucleus. Arrows, arrowheads and asterisks indicate vesiculated membrane structures, ER patches and dilated ER, respectiv...

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Section: Introductionmentioning

confidence: 99%

Role of syntaxin 18 in the organization of endoplasmic reticulum subdomains

Iinuma

Aoki

Arasaki

et al. 2009

Journal of Cell Science

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“…However, in this case, it was conceivable that the observed inhibition might have been caused by a failure of the cell to express Fc␥RIIA-FLAG on the surface. Previously, we reported that syntaxin 18 plays an important role in protein transport between the ER and the Golgi (Hatsuzawa et al, 2000), and we have recently shown that D12 is involved in post-Golgi transport (Okumura et al, 2006). This prompted us to investigate the cell surface expression of Fc␥RIIA-FLAG in 293T/RIIA-FLAG cells transfected with siRNA.…”

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confidence: 99%

“…This Q-SNARE and an abundant R-SNARE, Sec22b (Hay et al, 1997;Chao et al, 1999;Zhang et al, 1999), have been proposed to play a role in vesicular transport between the ER and Golgi (Zhang et al, 1999;Hatsuzawa et al, 2000). D12 (a mammalian homologue of yeast Use1p/Slt1p) is another Q-SNARE that associates with the ER and binds to both syntaxin18 and Sec22b (Okumura et al, 2006). Syntaxin18 is also known to associate with a wide variety of proteins, including ZW10, a spindle checkpoint protein .…”

mentioning

confidence: 99%

Involvement of Syntaxin 18, an Endoplasmic Reticulum (ER)-localized SNARE Protein, in ER-mediated Phagocytosis

Hatsuzawa

Tamura

Hashimoto

et al. 2006

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104

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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...

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“…Stxl8 on the other hand, like Stx5, has a 17 amino acid residue long transmembrane domain, which is indicative of an ER-Golgi membrane associated SNARE (Hatsuzawa et al 2000;Okumura et al 2006).…”

Section: Syntaxin 18 An Er-associated Q-snarementioning

confidence: 99%

“…Of special interest concerning the structure of Stxl8 is the short length of its transmembrane domain; most plasma-membrane located SNAREs, including StxlA and Stx2, have transmembrane domains >23 amino acid residues in length (Okumura et al 2006). Stxl8 on the other hand, like Stx5, has a 17 amino acid residue long transmembrane domain, which is indicative of an ER-Golgi membrane associated SNARE (Hatsuzawa et al 2000;Okumura et al 2006).…”

Section: Syntaxin 18 An Er-associated Q-snarementioning

confidence: 99%

Expression, purification and evaluation of recombinant human Syntaxin 18 as an endoribonuclease.

Bennett¹

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Involvement of a Novel Q-SNARE, D12, in Quality Control of the Endomembrane System

Cited by 27 publications

References 58 publications

Role of syntaxin 18 in the organization of endoplasmic reticulum subdomains

Role of syntaxin 18 in the organization of endoplasmic reticulum subdomains

Involvement of Syntaxin 18, an Endoplasmic Reticulum (ER)-localized SNARE Protein, in ER-mediated Phagocytosis

Expression, purification and evaluation of recombinant human Syntaxin 18 as an endoribonuclease.

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