Effect of siRNA for SNARE proteins on exocytosis from salivary glands

SNAP-23是具有2个串联SNARE基序（Qb和Qc）的SNAP-25的非神经元同种型，被认为在大多数不表达SNAP-25的细胞中作为t-SNARE对调节性和组成性胞吐作用起关键作用。在这里，我们评估了SNAP-23在HeLa细胞的组成性胞吐途径中的作用。尽管通过用针对SNAP-23的siRNA进行基因沉默，SNAP-23的水平降低至对照值的10%以下，但是当瞬时表达的SEAP（分泌的碱性磷酸酶）被测量为组成性分泌的标志物时，胞吐是正常的。SNAP-23和syntaxin-4的双敲低也未能抑制分泌。此外，δC8-SNAP-23（显性阴性SNAP-23）的过表达没有消除SEAP分泌。然而，当通过GFP标记的人生长激素（hGH-GFP）的表达来观察分泌途径时，在过表达δC8-SNAP-23的细胞的外周质膜中观察到含有hGH-GFP的囊泡的积累，尽管hGH-GFP的释放量没有像SEAP分泌的情况那样减少。这些结果表明，SNAP-23参与组成性胞吐，但不是必需的，至少对于分泌蛋白的简单胞吐。

SNAP-23, a non-neuronal isoform of SNAP-25 with 2 tandem SNARE motifs (Qb and Qc), is believed to play a key role as a t-SNARE for both regulated and constitutive exocytosis in most cells that do not express SNAP-25. Here we evaluated the role of SNAP-23 in constitutive exocytotic pathways of HeLa cells. Although the level of SNAP-23 was reduced to less than 10% of the control value by gene silencing with siRNA directed against SNAP-23, exocytosis was normal when transiently expressed SEAP (secreted alkaline phosphatase) was measured as a marker of constitutive secretion. Double knockdown of SNAP-23 and syntaxin-4 also failed to inhibit the secretion. Furthermore, over-expression of δC8-SNAP-23, a dominant-negative SNAP-23, did not abrogate SEAP secretion. When the secretory pathway was visualized by the expression of GFP-tagged human growth hormone (hGH-GFP), however, the accumulation of vesicles containing hGH-GFP was observed in the peripheral plasma membrane of the cells that over-expressed δC8-SNAP-23, although the quantity of hGH-GFP release was not decreased as in the case of SEAP secretion. These results suggest that SNAP-23 is involved in constitutive exocytosis, but is not essential at least for simple exocytosis of secretory proteins.