Department of Clinical Biochemistry, University of Cambridge, Cambridge Institute for Medical Research, Wellcome Trust/MRC Building, Hills Road, Cambridge CB20XY, UK.
Traffic. 2010 Sep;11(9):1191-204. doi: 10.1111/j.1600-0854.2010.01087.x. Epub 2010 Jun 10.
The role of SNAREs in mammalian constitutive secretion remains poorly defined. To address this, we have developed a novel flow cytometry-based assay for measuring constitutive secretion and have performed a targeted SNARE and Sec1/Munc18 (SM) protein-specific siRNA screen (38 SNAREs, 4 SNARE-like proteins and 7 SM proteins). We have identified the endoplasmic reticulum (ER)/Golgi SNAREs syntaxin 5, syntaxin 17, syntaxin 18, GS27, SLT1, Sec20, Sec22b, Ykt6 and the SM protein Sly1, along with the post-Golgi SNAREs SNAP-29 and syntaxin 19, as being required for constitutive secretion. Depletion of SNAP-29 or syntaxin 19 causes a decrease in the number of fusion events at the cell surface and in SNAP-29-depleted cells causes an increase in the number of docked vesicles at the plasma membrane as determined by total internal reflection fluorescence (TIRF) microscopy. Analysis of syntaxin 19-interacting partners by mass spectrometry indicates that syntaxin 19 can form SNARE complexes with SNAP-23, SNAP-25, SNAP-29, VAMP3 and VAMP8, supporting its role in Golgi to plasma membrane transport or fusion. Surprisingly, we have failed to detect any requirement for a post-Golgi-specific R-SNARE in this process.
SNAREs 在哺乳动物组成型分泌中的作用仍未得到明确界定。为了解决这个问题，我们开发了一种新的基于流式细胞术的测定方法来测量组成型分泌，并进行了靶向 SNARE 和 Sec1/Munc18 (SM) 蛋白特异性 siRNA 筛选（38 个 SNAREs、4 个 SNARE 样蛋白和 7 个 SM 蛋白）。我们已经确定了内质网 (ER)/高尔基体 SNAREs 突触素 5、突触素 17、突触素 18、GS27、SLT1、Sec20、Sec22b、Ykt6 和 SM 蛋白 Sly1，以及高尔基后 SNAREs SNAP-29 和突触素 19，它们是组成型分泌所必需的。SNAP-29 或突触素 19 的耗竭会导致细胞表面融合事件数量减少，而在 SNAP-29 耗竭的细胞中，在全内反射荧光 (TIRF) 显微镜下会导致质膜上停靠囊泡的数量增加。通过质谱分析突触素 19 的相互作用伙伴表明，突触素 19 可以与 SNAP-23、SNAP-25、SNAP-29、VAMP3 和 VAMP8 形成 SNARE 复合物，支持其在高尔基体到质膜运输或融合中的作用。令人惊讶的是，我们未能在此过程中检测到任何后高尔基特异性 R-SNARE 的需求。
