VAMP3/Syb and YKT6 are required for the fusion of constitutive secretory carriers with the plasma membrane.
VAMP3/Syb and YKT6 are required for the fusion of constitutive secretory carriers with the plasma membrane.
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DOI：
10.1371/journal.pgen.1006698
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发表时间：
2017-04
期刊：
影响因子：
4.5
通讯作者：
Peden AA
中科院分区：
文献类型：
--
作者：
Gordon DE;Chia J;Jayawardena K;Antrobus R;Bard F;Peden AA
后生动物中组成型分泌囊泡与质膜融合所需的细胞机制仍不明确。为解决这一问题，我们开发了一种强大的定量检测分泌的方法，并将其与果蝇细胞中的组合基因缺失研究相结合。这使我们能够鉴定出至少三种介导高尔基体到质膜运输的SNARE复合物（STX1、SNAP24/29和Syb；STX1、SNAP24/29和YKT6；STX4、SNAP24和Syb）。在哺乳动物细胞中，RNAi介导的YKT6和VAMP3缺失也会阻断组成型分泌，这表明YKT6在这一过程中具有进化上保守的作用。YKT6在质膜融合中意想不到的作用可能在一定程度上解释了为什么RNAi和基因破坏研究在高等真核生物中未能产生预期的表型。
组成型分泌途径将新合成的蛋白质和脂质输送到细胞表面，对细胞生长和生存至关重要。该途径是抗体、细胞因子和细胞外基质成分等分子分泌所必需的，因此具有重要的生理和商业意义。大多数分泌蛋白在内质网开始其旅程，经过高尔基体，并以小囊泡/小管的形式运输到细胞表面，然后与质膜融合。令人惊讶的是，对这一融合步骤的分子层面的理解仍然不清楚，而且在高等真核生物中，不知道哪些SNARE蛋白驱动这一过程。为解决这一问题，我们开发了一种强大的定量检测分泌的方法，并将其与果蝇细胞中的基因缺失研究相结合。利用这种检测方法，我们鉴定出三种驱动分泌囊泡与质膜融合的SNARE复合物，并揭示了R - SNARE YKT6在这一过程中意想不到的作用。利用这一知识，我们重新审视了SNARE在哺乳动物细胞中分泌载体与质膜融合中的作用，并发现YKT6在这一过程中具有进化上保守的作用。
The cellular machinery required for the fusion of constitutive secretory vesicles with the plasma membrane in metazoans remains poorly defined. To address this problem we have developed a powerful, quantitative assay for measuring secretion and used it in combination with combinatorial gene depletion studies in Drosophila cells. This has allowed us to identify at least three SNARE complexes mediating Golgi to PM transport (STX1, SNAP24/29 and Syb; STX1, SNAP24/29 and YKT6; STX4, SNAP24 and Syb). RNAi mediated depletion of YKT6 and VAMP3 in mammalian cells also blocks constitutive secretion suggesting that YKT6 has an evolutionarily conserved role in this process. The unexpected role of YKT6 in plasma membrane fusion may in part explain why RNAi and gene disruption studies have failed to produce the expected phenotypes in higher eukaryotes. The constitutive secretory pathway delivers newly synthesised proteins and lipids to the cell surface and is essential for cell growth and viability. This pathway is required for the secretion of molecules such as antibodies, cytokines and extracellular matrix components so has both significant physiological and commercial importance. The majority of secreted proteins begin their journey at the endoplasmic reticulum, pass through the Golgi, and are transported to the cell surface in small vesicles/tubules which fuse with the plasma membrane. Surprisingly, the molecular understanding of this fusion step is still unclear and in higher eukaryotes it is not known which SNARE proteins drive this process. To address this problem we have developed a powerful, quantitative assay for measuring secretion and used it in combination with gene depletion studies in Drosophila cells. Using this assay we identified three SNARE complexes driving the fusion of secretory vesicles with the plasma membrane and uncovered an unexpected role for the R-SNARE YKT6 in this process. Using this knowledge we have re-examined the role of SNAREs in the fusion of secretory carriers with the plasma membrane in mammalian cells and have found that YKT6 has an evolutionarily conserved role in this process.