Department of Orthodontics, School of Dentistry, Health Sciences University of Hokkaido, Tobetsu, Hokkaido 061-0293, Japan.
Mol Cell Biochem. 2012 Jul;366(1-2):159-68. doi: 10.1007/s11010-012-1293-z. Epub 2012 Apr 3.
To evaluate the role of SNARE proteins in the constitutive exocytosis, we knocked down syntaxin 3, 4, 5, 6, 7, and VAMP3, 5, 7, 8 with their siRNAs, and determined the cell-to-medium ratio of CLuc, a secreted luciferase of Cypridina noctiluca. Although the protein level of SNAREs in HeLa cells was markedly reduced by the siRNA treatment, the cell/medium ratio was scarcely increased by any siRNAs except for syntaxin 5. The accumulation of GFP-tagged human growth hormone was also visible only by the knockdown of syntaxin 5. To examine whether the residual amount of SNAREs are sufficient for maintaining normal constitutive exocytosis, we estimated the effect of siRNAs on the level of post-Golgi SNARE complexes containing syntaxin 4, SNAP23, and VAMP3 or VAMP8. The amount of SNARE complexes was robustly decreased by siRNAs and was well correlated with the residual amount of SNAREs in the lysates, suggesting that SNAREs are unnecessarily excessive for the formation of post-Golgi SNARE complexes in HeLa cells.
为了评估 SNARE 蛋白在组成型胞吐作用中的作用，我们用它们的 siRNA 敲低了突触素 3、4、5、6、7 和 VAMP3、5、7、8，并用萤光素酶报告基因检测了细胞向培养基中分泌的 CLuc。虽然 SNARE 蛋白的水平在 HeLa 细胞中经 siRNA 处理后显著降低，但除了突触素 5 之外，任何 siRNA 都没有显著增加细胞/培养基的比值。只有敲低突触素 5 才能观察到 GFP 标记的人生长激素的积累。为了研究剩余 SNARE 蛋白的数量是否足以维持正常的组成型胞吐作用，我们评估了 siRNA 对含有突触素 4、SNAP23 和 VAMP3 或 VAMP8 的高尔基后 SNARE 复合物水平的影响。SNARE 复合物的数量因 siRNA 而显著减少，并且与裂解物中剩余 SNARE 蛋白的数量呈良好相关性，这表明在 HeLa 细胞中，SNARE 蛋白对于形成高尔基后 SNARE 复合物是不必要的过量。
