Effect of siRNA for SNARE proteins on exocytosis from salivary glands

Snap-23是SNAP-25的一个非神经元亚型，具有两个串联的SNARE基序(QB和QC)，被认为在大多数不表达SNAP-25的细胞中扮演着调节和结构性胞吐的t-陷阱的关键角色。在这里，我们评估了SNAP-23在HeLa细胞构件性胞吐途径中的作用。用针对SNAP-23的siRNA进行基因沉默后，SNAP-23的表达水平下降到对照水平的10%以下，但当瞬时表达的分泌型碱性磷酸酶(SEAP)被检测为结构性分泌的标志时，胞吐作用是正常的。SNAP-23和Synaxin-4的双重敲除也不能抑制这种分泌。此外，δC8-SNAP-23的过度表达并没有抑制SEAP的分泌。然而，当通过绿色荧光蛋白标记的人生长激素(hGH-GFP)的表达来显示分泌途径时，尽管hGH-GFP的释放量并没有像δ分泌时那样减少，但在高表达SEAPC8-SNAP-23的细胞的外周质膜上观察到了含有hGH-GFP的囊泡的聚集。这些结果表明，SNAP-23参与了结构性胞吐作用，但至少对简单的分泌蛋白胞吐作用不是必需的。

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.