• Mitochondrial E3 Ubiquitin Ligase MARCH5 Coordinates Mitochondrial Membrane Dynamics in the Outer Mitochondrial Membrane Associated Degradation (OMMAD) Pathway

      Das, Shweta; Karbowski, Mariusz (2015)
      The molecular mechanisms of the pathway governing mitochondrial and cellular homeostasis via outer mitochondrial membrane (OMM)- associated degradation (OMMAD) are not clear. We found that stability of OMM proteins MiD49, a mitochondrial fission factor, and Mcl1, an anti-apoptotic Bcl-2 family protein, is controlled by OMM-associated E3 ubiquitin ligase MARCH5. Accumulation of MiD49 and Mcl1, but not other proteins, and selective inhibition of proteasome-dependent degradation was seen in MARCH5 knockout cells. Through immunofluorescence, mitochondrial fusion, and biochemical assays, we found that MARCH5 governs Mcl1 turnover indirectly, but regulates MiD49 directly through ubiquitination. Furthermore, factors inducing stress-dependent apoptosis and mitochondrial toxins induced MARCH5-dependent MiD49 degradation, showing MARCH5 control of MiD49 stability as a novel stress response mechanism. Accordingly, MARCH5 depleted cells were more sensitive to stress-induced apoptosis. These findings provided evidence supporting a central role of MARCH5 in Mcl1 and MiD49 turnover and coordination of the OMMAD pathway with the mitochondrial and cellular stress response.
    • Novel Signaling Mechanisms in the Regulation of Mitochondrial Dynamics

      Cherok, Edward Patrick, Jr.; Karbowski, Mariusz (2017)
      Mitochondria are dynamic organelles that constantly undergo fission and fusion events (referred to as mitochondrial dynamics) to form highly interconnected networks within cells. These networks allow mitochondria to share resources such as mitochondrial DNA and antioxidant molecules to maintain the health of the network. Because mitochondria are the main source of production of ATP through oxidative phosphorylation, and also regulate cell death through apoptosis, it is critically important to maintain homeostasis in these organelles. Indeed, dysfunction in mitochondrial dynamics has been linked to numerous diseases, including cancer, neurodegenerative, endocrine, and cardiovascular diseases. Therefore, understanding the mechanisms by which mitochondrial dynamics contributes to the overall health of this organelle is of great interest. The primary proteins involved in the regulation of mitochondrial fusion and fission, and the mechanisms by which they act, are generally understood. It is also well accepted that mitochondrial fusion and fission is balanced; however, how these two separate processes communicate and signal to each other is currently unknown. To better understand the crosstalk between mitochondrial fission and fusion, we studied a function of the outer mitochondrial membrane associated E3 ubiquitin ligase, MARCH5. We found that MARCH5 acts as a negative regulator of mitochondrial fission through the ubiquitin-dependent degradation of the fission factor, MiD49. Shedding light on a possible mechanism by which the activities of fission factors are coordinated, we found that the Drp1 receptor, Mff, promotes MiD49 stability by negatively regulating MARCH5 activity, thereby enhancing mitochondrial fission rates. Finally, supporting molecular crosstalk between fission and fusion, we found that Mff also regulates the stability of the outer mitochondrial membrane fusion factors, Mfn1 and Mfn2, and that loss of Mff expression/activity results in reduced mitochondrial fusion rates in those cells. Thus, the studies presented here display novel crosstalk and signaling mechanisms by which fission factors are able to fine-tune mitochondrial fission and fusion rates through modification of the ubiquitin-proteasome system.