Department of Cell and Molecular Biology, Biomedicum, Karolinska Institutet, Solnavägen 9, 17177, Stockholm, Sweden.
Sci Rep. 2020 Dec 18;10(1):22334. doi: 10.1038/s41598-020-78845-7.
Even though K63-linked polyubiquitin chains do not target proteins for proteasomal degradation, they play nevertheless a complementary protective role in maintaining protein homeostasis by directing malfunctioning proteins and organelles to inclusion bodies or autophagosomes. A paradigm for this process is the sequestration and autophagic degradation of dysfunctional mitochondria. Although studies have shown that K63-ubiquitylation of mitochondrial proteins by the ubiquitin ligase Parkin is important in this process, it is presently not clear if this modification also suffices to initiate this cascade of events. To address this question, we have engineered the ubiquitin ligase ProxE3, which in an inducible manner synthesizes K63-linked ubiquitin chains on the surface of mitochondria. We found that the presence of K63-linked ubiquitin chains on mitochondria resulted in the recruitment of the ubiquitin adaptor p62 and induced a dramatic redistribution of mitochondria, which was reminiscent to the Parkin-facilitated sequestration in response to mitochondrial uncoupler. However, ProxE3 did not induce autophagic degradation of mitochondria. Our data show that K63-linked ubiquitin chains at the mitochondrial membrane are sufficient for the induction of mitochondrial sequestration, but not mitophagy, without the need of extrinsically inflicting mitochondrial dysfunction.
尽管 K63 连接的多泛素链不会将蛋白质靶向蛋白酶体降解，但它们通过将功能失调的蛋白质和细胞器导向包含体或自噬体，在维持蛋白质平衡方面发挥着互补的保护作用。这个过程的一个范例是功能失调的线粒体的隔离和自噬降解。尽管研究表明，泛素连接酶 Parkin 对线粒体蛋白的 K63 泛素化在这个过程中很重要，但目前尚不清楚这种修饰是否足以启动这一连串事件。为了解决这个问题，我们设计了泛素连接酶 ProxE3，它以诱导的方式在线粒体表面合成 K63 连接的泛素链。我们发现，线粒体上 K63 连接的泛素链的存在导致了泛素接头 p62 的募集，并诱导了线粒体的剧烈重分布，这让人联想到线粒体解偶联剂诱导的 Parkin 促进的隔离。然而，ProxE3 并没有诱导线粒体的自噬降解。我们的数据表明，线粒体膜上的 K63 连接的泛素链足以诱导线粒体的隔离，但不会诱导线粒体自噬，而不需要外在地引起线粒体功能障碍。
