Centre for Mechanochemical Cell Biology and Division of Biomedical Cell Biology, Warwick Medical School, University of Warwick, Coventry, United Kingdom.
Elife. 2022 Jul 19;11:e78929. doi: 10.7554/eLife.78929.
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.
网格蛋白包被小泡(CCV)的形成是一种主要的膜重塑过程,对细胞内的膜运输至关重要。除了网格蛋白,这些小泡还包含至少 100 种不同的蛋白质,尽管不清楚有多少种对小泡的形成是必不可少的。在这里,我们表明,使用最小的机制可以在活细胞中诱导细胞内网格蛋白包被的形成,并且可以在各种膜上实现,包括线粒体的外膜。化学异二聚化被用来将一个网格蛋白结合片段“钩子”诱导性地附着到一个靶向特定膜的“锚”蛋白上。内源性网格蛋白在诱导后几秒钟内组装到线粒体上形成被称为 MitoPits 的包被陷窝,MitoPits 是双层内陷,优先形成在线粒体的高曲率区域。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个过程中的功能参与的证据。此外,MitoPit 衍生小泡的分裂独立于已知的分裂因子,包括动力蛋白和动力相关蛋白 1(Drp1),这表明网格蛋白笼产生足够的力来出芽细胞内小泡。我们的结果表明,在被招募后,网格蛋白足以形成细胞内的 CCV。