y between cells and even within a cell, depending on the metabolic states. Cardiomyocytes and neurons, due to their high energy demands, have greater amounts. Mitochondria are dynamic and form reticular networks to assure efficient distribution of mitochondrial DNA (mtDNA) and proteins. The fission and fusion of mitochondria are features of mitochondria dynamics along with their trafficking along microtubule routes in higher organisms (actin in yeast). Mitochondria dynamics and the mitochondrial autophagy (mitophagy) pathways are intimately connected. Fission is believed to be a pre-requisite for mitophagy, allowing for the delineation of individual, damaged mitochondria while mitophagy targets components of the fusion apparatus and of transport for degradation. Of the ~1,500 mitochondrial proteins, only 13 are encoded in the mtDNA, the rest (~99%), have to be imported. As such, the mitochondrial protein import pathway impacts on every aspect of mitochondrial function, including mitochondria dynamics and autophagy. Mitochondrial autophagy is a specialized aspect of the overall, non-selective bulk macroautophagy, or autophagy pathway (click to link to the diagram page). Autophagy provides nutrients under starvation conditions and remove stress damaged proteins and organelles. Mitophagy allows for the removal of damaged mitochondria and for mitochondrial depletion, as required in certain cell states or in development. The mitochondrial autophagy pathway is presented here. Removal of mitochondria proceeds via alternative routes. One involves the Pink1-Parkin duo responding to loss of mitochondrial membrane potential and mediated via ubiquitination events. Another route involves the mitophagy receptors and responds to stresses such as hypoxia or facilitates mitochondria removal under normal physiological, developmental conditions. Mitophagy shares elements with bulk autophagy but also exhibits unique elements whose detailed molecular functions and their regulation, are still to be determined. In all cases, damaged mitochondria are targeted to an autophagosome at a stage subsequent to its initiation. Largely, the upstream elements are not well known, although autophagy components such as Beclin1 or Ulk1, have been shown to be required for mitophagy. Mitochondrial stress also promotes the translocation of cardiolipin (CL), the signature phospholipid of IMM, too OMM; the externalized CL will trigger mitophagy or apoptosis depending on the magnitude of the injury.

Pink1-Parkin
Pink1-Parkin is the main route of mitochondria quality control (QC) involving the nuclear-encoded mitochondrial serine/threonine kinase Pink1 and downstream of it, the cytosolic E3 ubiquitin ligase Parkin. Mutations in these two genes have been associated with the familial form of the neurodegenerative condition Parkinson Disease (PD). Pink1 is proposed to acts as a mitochondrial stress sensor. Under normal conditions Pink1 is imported to mitochondria via the outer mitochondrial membrane (OMM) and inner mitochondrial membrane (IMM) translocators, TOM and TIM, respectively. In the IMM, Pink1 is cleaved by proteases such as PARL and subsequently degraded. When the mitochondrial membrane potential is dissipated, Pink1 translocation to IMM is abolished and Pink1 accumulates to OMM where is further stabilized by the TOMM7 component of TOM. The kinase activity promotes recruitment of Parkin to the damaged mitochondria and is necessary for its E3 ubiquitin ligase activity. Parkin ubiquitinates itself and substrates and the ubiquitin conjugates can be phosphorylated by Pink1 to further increase parkin activity in a feed-forward loop. Some of the ubiquitinated (Ub) substrates are targeted for proteasomal degradation such as mitofusin1 and 2, the two GTPases of the mitochondrial fusion arm and the Miro GTPases in the transport network in the mitochondria dynamics pathway (Miro is also substrate for Pink1 phosphorylation). Inhibition of fusion helps fragment mitochondria networks and isolate damaged mitochondria via fission, and also precludes acquisition of damaged mitochondria within networks ; inhibition of transport will prevent delivery of damaged mitochondria at sites of action while promoting a retrograde transport for degradation. Also a Parkin substrate, is the transcriptional regulator Znf746, known as Paris - a repressor of Pgc-1alpha transcriptional co-activator regulating the expression of genes important for mitochondria function. Other Ub-OMM proteins will promote the recruitment of Ub-binding autophagy receptors/adapters such as Sqstm1/p62 and optineurin. These proteins contain a ubiquitin-associated domain (UBA) and an LC3-interacting region (LIR). The mechanisms and implications of these proteins in mitophagy are still to be fully elucidated, but they can link Pink1-Parkin ubiquitination to the autophagosome, and thus mitochondrial autophagy. LC3 proteins and homologs are components of the second conjugation-like LC3 system in the elongation step of autophagy. The system is also required for the closure of the autophagosome which will then lead to its maturation, fusion with the lysosome and degradation of the cargo, here mitochondria. Ubiquitination, like many of the post-translational modifications is made reversible through the action of deubiquitinases (DUBs) which regulate E3 ligase function. DUBs that target Parkin include ataxin-3 and Usp8, while those that target Parkin substrates include Usp15 and Usp30. Of note is that Atxn3 acts via binding the E2-Ub-cojugating enzyme Ube2l3 and thus preventing Parkin self-ubiquitination.

Mitophagy receptors
The mitophagy receptors are involved in the removal of mitochondria as reticulocytes transform into mature red blood cells and of sperm-derived mitochondria in fertilized oocytes, both programmed processes. This route also elicits mitochondrial autophagy in response to hypoxia-induced stress. The receptors include Bnip3 and Bnip3l (known as Nix) and Fundc1. All three OMM resident proteins possess an LC3-interacting region (LIR) that allows them to target mitochondria to the autophagosome. Bnip3 and Bnip3l are members of the BH3-only Bcl proteins with a role in the intrinsic apoptotic pathway where they bind to and neutralize the anti-apoptotic proteins. The promoters of Bnip3 and Bnip3l contain hypoxia response elements (HRE), the binding site for the hypoxia inducible factor heterodimer. Hypoxia inducible factor pathway is considered the master regulator of oxygen homeostasis. The heterodimer is composed of the constitutive beta factor and the alpha factor which, under normoxic conditions, is targeted for degradation in an oxygen-dependent manner. Oxygen is the terminal acceptor of electrons in the electron transport chain (ETC) arm of OXPHOS pathway and hypoxia is detrimental to mitochondria as well as to cells, overall. Both mitophagy and bulk autophagy can be triggered by hypoxia. Mechanistically, Fundc1-dependent mitophagy in response to hypoxia appears to be dependent on its phosphorylation state. Under normoxic conditions Fundc1 is phosphorylated by Src and casein kinase 2 (CK2) complex. Hypoxia releases an inhibitory effect on the Pgam5 phosphatase; the dephosphorylated Fundc1 has a much greater affinity for LC3.

Cardiolipin
Cardiolipin (CL) is the signature phospholipid of the IMM. Its translocation to OMM, which can also occur during its remodeling, serves as a 'signal' of mitochondrial injury. Depending on the magnitude of the signal, it will trigger mitophagy or apoptosis; mitophagy does not require modification of CL whereas apoptosis necessitates both the presence of CL in the OMM and its oxidative modification, specifically peroxidation. The externalized CL interacts with LC3 components of the LC3 system. Deletion of putative CL binding sites in these components abolish CL-mediated mitophagy. Molecular modeling was based on the crystal structure of Map1lc3b. Mitochondria damage and dysfunction are associated with a range of conditions including neurodegenerative diseases and with ageing. To see the ontology report for annotations, GViewer and download, click here...(less)