Summary
Mitochondria are at the center of age-related human diseases, such as Parkinson’s, Alzheimer’s, Huntington’s
diseases. With current trends toward the aging population, developing therapies for these diseases is a critical
need. Protective strategies, mostly aimed at neutralizing toxic reactive oxygen species (ROS), have only been
partially successful. Thus, new treatment options with independent modes of action are needed to improve the
success rate of current therapies. The proposed work directly responds to this need and will provide critical
insights into the regulation and function of mitochondrial quality control pathways. Eukaryotic cells have a
multi-layered system dedicated to mitochondrial quality control. The initial defense consists of a network of
proteolytic systems that degrade proteins that are damaged, misfolded or mislocalized, or control activities of
stress responsive mitochondrial factors. Recent findings, including work in the PI’s laboratory, demonstrated
that the ubiquitin (Ub)/proteasome system (UPS), through Ub-dependent degradation/control of the OMM
associated proteins is critical for mitochondrial quality control. Regulation of mitochondrial fusion and fission
(mitochondrial dynamics) is another principal mitochondrial quality control mechanism. Recently we proposed
that stability of outer mitochondrial membrane (OMM) proteins, including mitochondrial fusion factors Mfn1 and
Mfn2 is controlled by mitochondrial fission proteins Drp1 and Mff. Consistent with this, our new data indicate
that previously unsuspected Ub-dependent signaling functions of Drp1 and Mff are required for this process.
We also found that mitochondrial protein ubiquitination is focal and that this process is accelerated in Mff-/- and
Drp1-/- cells. These and other preliminary results, suggest unappreciated mechanisms regulating and
coordinating mitochondrial quality control pathways, including participation of the novel mitochondrial protein
degradation intermediates and mutual dependence between mitochondrial dynamics and mitophagy. We also
identified an OMM-associated E3 Ub ligase Rnf179, which according to our preliminary findings not only
controls stability of OMM proteins, including Mfn1 and Mfn2, but also affects mitochondria-specific autophagy
(mitophagy), a critical UPS-dependent mitochondrial quality control pathway. The principle goal of this proposal
is to test the hypothesis that through Ub-dependent signaling, Drp1 and Mff control and coordinate distinct
mitochondrial quality pathways, including mitochondrial fission and fusion rates and activation of mitophagy.
We also anticipate defining the role and mechanism of Rnf179 with mitochondrial protein ubiquitination in the
control of mitochondrial function and integration of mitochondrial quality control pathways, including Parkin-
dependent and -independent mitophagy. To achieve these goals we combine biochemical, state-of-the-art
imaging, and recently developed gene editing methods to address the following questions: (1) What is the Ub-
dependent mechanism that mediates crosstalk between mitochondrial fission and fusion machineries, and how
does it maintain balance between these two processes? (2) What is the mechanism by which Rnf179 in
concert with Mff and Drp1 controls mitochondrial homeostasis? (3) What is the mechanism by which Rnf179 in
concert with Mff and Drp1 complex controls mitophagy?