Role of syntaxin 18 in the organization of endoplasmic reticulum subdomains

Abstract

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...

<b>Changes in the three-dimensional ultrastructure of membranous organelles in male rat pituitary gonadotropes after castration</b>

“…The ultrastructure of the anomalous tubuloreticular ER cluster demonstrated in the present study rather resembles that of the random tubular ER patches described in recent two studies (20,47). One of these two studies revealed anomalous ER patches in HeLa cells in which an ER-related soluble NSF attachment protein receptor (SNARE) protein, syntaxin 18, is depleted by knockdown with specific siRNAs (20).…”

“…One of these two studies revealed anomalous ER patches in HeLa cells in which an ER-related soluble NSF attachment protein receptor (SNARE) protein, syntaxin 18, is depleted by knockdown with specific siRNAs (20). Syntaxin 18 is localized in the ER, and is thought to be involved in retrograde transport from the Golgi to the ER as well as homotypic ER membrane fusion (17).…”

“…Syntaxin 18 is localized in the ER, and is thought to be involved in retrograde transport from the Golgi to the ER as well as homotypic ER membrane fusion (17). Knockdown of syntaxin 18 also induces dispersion of ERGIC and the Golgi apparatus, in addition to the occurrence of ER patches (20). The mechanisms and physiological significance behind the formation of the anomalous ER patches/ clusters and disorganization of the Golgi apparatus are largely obscure, both in the stimulated gonadotropes and syntaxin 18-knockdown HeLa cells.…”

Contribution of the long form of syntaxin 5 to the organization of the endoplasmic reticulum

“…Depletion of Syn18 causes remarkable aggregation of the smooth ER membrane, although rough ER membranes are relatively intact (Iinuma et al, 2009). Knockdown of BNIP1 resulted in the loss of the three-way junction of the peripheral ER (Nakajima et al, 2004).…”

Interaction of α-taxilin Localized on Intracellular Components with the Microtubule Cytoskeleton

“…3C). Finally, syntaxin-18, a syntaxin member resided on the ER, was depleted in Hela cells by introducing siRNA oligo duplex against syntaxin-18 as previously described (Iinuma et al, 2009). When the treated cells were roughly fractionated to the cytosol and membrane fractions, depletion of syntaxin-18 hardly affected the subcellular distribution of α-taxilin (Fig.…”