Abstract
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.
Highlights
Even though K63-linked polyubiquitin chains do not target proteins for proteasomal degradation, they play a complementary protective role in maintaining protein homeostasis by directing malfunctioning proteins and organelles to inclusion bodies or autophagosomes
We investigated the proficiency of K63-linked polyubiquitin chains to target mitochondria for sequestration and autophagy
We developed a ubiquitin ligase that decorated an inherent reference protein located at the outer membrane of mitochondria with K63-linked polyubiquitin chains
Summary
Even though K63-linked polyubiquitin chains do not target proteins for proteasomal degradation, they play a complementary protective role in maintaining protein homeostasis by directing malfunctioning proteins and organelles to inclusion bodies or autophagosomes. The ringbetween-ring (RBR) ubiquitin ligase Parkin translocates to depolarized mitochondria and decorates the dysfunctional mitochondria with K63-linked ubiquitin chains by modifying a number of proteins in the mitochondrial outer membrane (MOM)[8] This results in the recruitment of the ubiquitin receptor p62 that bridges the mitochondria to the dynein transport machinery facilitating retrograde transport to aggresome-like structures in the perinuclear r egion[6,9]. Parkin-mediated ubiquitylation of mitochondrial proteins appears to play a dual role since it regulates the OPTN/NDP52-facilitated targeting of mitochondria for autophagy[11], independent from its ability to stimulate p62-dependent targeting of mitochondria for retrograde transport[15,16] It is, presently unclear if K63-linked ubiquitin chains are sufficient to initiate this process as, in the context of damaged mitochondria, this ubiquitin mark is part of a more complex response involving a number of post-translational modifications. This process may promote sequestration and/or autophagy by preventing fusion of dysfunctional mitochondria[23], and exposing mitochondrial inner membrane (MIM) p roteins[24,25]
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