Lee Miriam, Ko Young-Joon, Moon Yeojin, Han Minsoo, Kim Hyung-Wook, Lee Sung Haeng, Kang KyeongJin, Jun Youngsoo
School of Life Sciences, Gwangju Institute of Science and Technology, Gwangju 500-712, Korea Integrative Aging Research Center, Gwangju Institute of Science and Technology, Gwangju 500-712, Korea Cell Dynamics Research Center, Gwangju Institute of Science and Technology, Gwangju 500-712, Korea.
School of Life Sciences, Gwangju Institute of Science and Technology, Gwangju 500-712, Korea Integrative Aging Research Center, Gwangju Institute of Science and Technology, Gwangju 500-712, Korea.
J Cell Biol. 2015 Aug 3;210(3):451-70. doi: 10.1083/jcb.201501043. Epub 2015 Jul 27.
Dynamin-like GTPases of the atlastin family are thought to mediate homotypic endoplasmic reticulum (ER) membrane fusion; however, the underlying mechanism remains largely unclear. Here, we developed a simple and quantitative in vitro assay using isolated yeast microsomes for measuring yeast atlastin Sey1p-dependent ER fusion. Using this assay, we found that the ER SNAREs Sec22p and Sec20p were required for Sey1p-mediated ER fusion. Consistently, ER fusion was significantly reduced by inhibition of Sec18p and Sec17p, which regulate SNARE-mediated membrane fusion. The involvement of SNAREs in Sey1p-dependent ER fusion was further supported by the physical interaction of Sey1p with Sec22p and Ufe1p, another ER SNARE. Furthermore, our estimation of the concentration of Sey1p on isolated microsomes, together with the lack of fusion between Sey1p proteoliposomes even with a 25-fold excess of the physiological concentration of Sey1p, suggests that Sey1p requires additional factors to support ER fusion in vivo. Collectively, our data strongly suggest that SNARE-mediated membrane fusion is involved in atlastin-initiated homotypic ER fusion.
Atlastin家族的动力蛋白样GTP酶被认为介导内质网（ER）同型膜融合；然而，其潜在机制仍不清楚。在此，我们开发了一种简单且定量的体外测定方法，使用分离的酵母微粒体来测量酵母Atlastin Sey1p依赖性内质网融合。通过该测定方法，我们发现内质网SNARE蛋白Sec22p和Sec20p是Sey1p介导的内质网融合所必需的。同样，Sec18p和Sec17p的抑制显著降低了内质网融合，Sec18p和Sec17p调节SNARE介导的膜融合。Sey1p与另一种内质网SNARE蛋白Sec22p和Ufe1p的物理相互作用进一步支持了SNARE蛋白参与Sey1p依赖性内质网融合。此外，我们对分离微粒体上Sey1p浓度的估计，以及即使Sey1p蛋白脂质体的浓度超过生理浓度25倍仍缺乏融合，表明Sey1p在体内需要其他因子来支持内质网融合。总体而言，我们的数据强烈表明SNARE介导的膜融合参与了Atlastin引发的内质网同型融合。
