Date Published: May 21, 2019
Publisher: Public Library of Science
Author(s): Laura Lee, Richard Seager, Yasuko Nakamura, Kevin A. Wilkinson, Jeremy M. Henley, David Chau.
The mitochondrial outer membrane protein Mitochondrial Fission Factor (Mff) plays a key role in both physiological and pathological fission. It is well established that at stressed or functionally impaired mitochondria, PINK1 recruits the ubiquitin ligase Parkin which ubiquitinates Mff and other mitochondrial outer membrane proteins to facilitate the removal of defective mitochondria and maintain the integrity of the mitochondrial network. Here we show that, in addition to this clearance pathway, Parkin also ubiquitinates Mff in a PINK1-dependent manner under non-stressed conditions to regulate constitutive Mff turnover. We further show that removing Parkin via shRNA-mediated knockdown does not completely prevent Mff ubiquitination under these conditions, indicating that at least one other ubiquitin ligase contributes to Mff proteostasis. These data suggest that that Parkin plays a role in physiological maintenance of mitochondrial membrane protein composition in unstressed cells through constitutive low-level activation.
Mitochondria are double membrane-bound organelles that generate 90% of cellular ATP . In most cells, mitochondria form extensive and dynamic networks, undergoing continuous cycles of fission and fusion. This creates a highly adaptable and efficient energy transfer system to rapidly deliver ATP to where it is most needed . In addition, fission plays a central role in the sequestration and selective degradation of defective mitochondria by mitophagy [3, 4]. Mitochondrial fission and fusion are both tightly regulated processes that are largely orchestrated by GTPases. Dynamin and dynamin-related protein (Drp) mediate fission [5, 6] whereas fusion of the mitochondrial outer and inner membranes is driven by the GTPases mitofusins (Mfn) 1 and 2 and Opa1, respectively .
Our data show that under basal conditions endogenous Parkin ubiquitinates Mff at K151. For this ubiquitination and subsequent Mff degradation Parkin needs to be activated by PINK1-dependent phosphorylation at S65. This Parkin-mediated ubiquitination of Mff coincides with Parkin-mediated Mff degradation, suggesting that Mff turnover is regulated by a Parkin-dependent, ubiquitin-mediated pathway. Mff is not a substrate of K48-linked ubiquitination but is a substrate of K63-linked ubiquitination. Furthermore, inhibition of the lysosome, but not the proteasome, rescues Mff from Parkin-mediated degradation. These data support a model in which Parkin-mediated degradation of Mff occurs via K63-linked ubiquitination and the lysosome. Interestingly, this activity appears to be in addition to mitophagy, in which depolarised mitochondria recruit Parkin to indiscriminately ubiquitinate MOM proteins prior to their degradation. Of the five MOM proteins assayed in Parkin-overexpressing cells, only Mff and, to a lesser extent, Fis1 were significantly rescued from degradation by inhibition of the lysosome (Fis1 was also rescued by proteasomal inhibition). Mfn2, Mid49 and VDAC were not significantly changed, despite Mfn2 and VDAC being known targets of CCCP-induced, Parkin-mediated mitophagy [32, 40, 41]. These data may indicate that mitophagy is not solely responsible for the changes we observe. Thus, we propose that Parkin may have a selective effect on the turnover of Mff, in addition to its role in mitophagy-dependent Mff degradation. The degradation of Mff by Parkin under basal conditions, together with its inactivity toward other known substrates under the same conditions, suggest that Parkin-mediated degradation of Mff is a regulatory mechanism independent of stress-dependent mechanisms. Moreover, since PINK1 is maintained at low levels in the MOM of healthy mitochondria, we propose that this mechanism plays a critical background role in maintaining mitochondrial integrity in the absence of induced stress.