Recruitment of clathrin to intracellular membranes is sufficient for vesicle formation.
Recruitment of clathrin to intracellular membranes is sufficient for vesicle formation.
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DOI：
10.7554/elife.78929
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发表时间：
2022-07-19
期刊：
影响因子：
7.7
通讯作者：
Royle, Stephen J.
中科院分区：
文献类型：
--
作者：
Kuey, Cansu;Sittewelle, Meghane;Larocque, Gabrielle;Hernandez-Gonzalez, Miguel;Royle, Stephen J.
网格蛋白包被囊泡（CCV）的形成是一个主要的膜重塑过程，对细胞内的膜运输至关重要。除了网格蛋白，这些囊泡含有至少100种不同的蛋白质，尽管目前还不清楚有多少是囊泡形成所必需的。在这里，我们表明，细胞内网格蛋白涂层的形成可以诱导活细胞中使用最小的机械，它可以实现各种膜，包括线粒体外膜。化学异源二聚化用于诱导性地将网格蛋白结合片段“钩”连接到靶向特定膜的“锚”蛋白。内源性网格蛋白在诱导后数秒内组装形成线粒体上的包被凹坑，称为MitoPits。MitoPit是双膜内陷，优先形成于线粒体的高曲率区域。在诱导后，CCV形成的所有阶段-起始，内陷，甚至分裂-都被忠实地重建。我们没有发现辅助蛋白在这一过程中的功能参与的证据。此外，分裂的MitoPit衍生的囊泡是独立的已知的断裂因子，包括发动蛋白和发动蛋白相关蛋白1（Drp 1），这表明网格蛋白笼产生足够的力芽细胞内囊泡。我们的研究结果表明，在其招聘，网格蛋白是足够的细胞内CCV的形成。
The formation of a clathrin-coated vesicle (CCV) is a major membrane remodeling process that is crucial for membrane traffic in cells. Besides clathrin, these vesicles contain at least 100 different proteins although it is unclear how many are essential for the formation of the vesicle. Here, we show that intracellular clathrin-coated formation can be induced in living cells using minimal machinery and that it can be achieved on various membranes, including the mitochondrial outer membrane. Chemical heterodimerization was used to inducibly attach a clathrin-binding fragment ‘hook’ to an ‘anchor’ protein targeted to a specific membrane. Endogenous clathrin assembled to form coated pits on the mitochondria, termed MitoPits, within seconds of induction. MitoPits are double-membraned invaginations that form preferentially on high curvature regions of the mitochondrion. Upon induction, all stages of CCV formation – initiation, invagination, and even fission – were faithfully reconstituted. We found no evidence for the functional involvement of accessory proteins in this process. In addition, fission of MitoPit-derived vesicles was independent of known scission factors including dynamins and dynamin-related protein 1 (Drp1), suggesting that the clathrin cage generates sufficient force to bud intracellular vesicles. Our results suggest that, following its recruitment, clathrin is sufficient for intracellular CCV formation.