Gorur Amita, Yuan Lin, Kenny Samuel J, Baba Satoshi, Xu Ke, Schekman Randy
Department of Molecular and Cell Biology and Howard Hughes Medical Institute, University of California, Berkeley, Berkeley, CA 94720.
Howard Hughes Medical Institute, University of California, Berkeley, Berkeley, CA 94720.
J Cell Biol. 2017 Jun 5;216(6):1745-1759. doi: 10.1083/jcb.201702135. Epub 2017 Apr 20.
The coat protein complex II (COPII) is essential for the transport of large cargo, such as 300-nm procollagen I (PC1) molecules, from the endoplasmic reticulum (ER) to the Golgi. Previous work has shown that the CUL3-KLHL12 complex increases the size of COPII vesicles at ER exit sites to more than 300 nm in diameter and accelerates the secretion of PC1. However, the role of large COPII vesicles as PC1 transport carriers was not unambiguously demonstrated. In this study, using stochastic optical reconstruction microscopy, correlated light electron microscopy, and live-cell imaging, we demonstrate the existence of mobile COPII-coated vesicles that completely encapsulate the cargo PC1 and are physically separated from ER. We also developed a cell-free COPII vesicle budding reaction that reconstitutes the capture of PC1 into large COPII vesicles. This process requires COPII proteins and the GTPase activity of the COPII subunit SAR1. We conclude that large COPII vesicles are bona fide carriers of PC1.
II型被膜小泡蛋白复合体（COPII）对于将大型货物（如300纳米的原胶原蛋白I（PC1）分子）从内质网（ER）运输到高尔基体至关重要。先前的研究表明，CUL3-KLHL12复合体可使内质网出口位点处的COPII小泡尺寸增加至直径超过300纳米，并加速PC1的分泌。然而，大型COPII小泡作为PC1运输载体的作用尚未得到明确证实。在本研究中，我们使用随机光学重建显微镜、关联光电子显微镜和活细胞成像技术，证明了存在可移动的COPII被膜小泡，这些小泡完全包裹货物PC1，并与内质网在物理上分离。我们还开发了一种无细胞COPII小泡出芽反应，该反应可将PC1捕获到大型COPII小泡中。这一过程需要COPII蛋白和COPII亚基SAR1的GTP酶活性。我们得出结论，大型COPII小泡是PC1的真正载体。
