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Mitochondrial E3 ubiquitin ligase MARCH5 controls mitochondrial fission and cell sensitivity to stress-induced apoptosis through regulation of MiD49 protein
Ubiquitin- and proteasome-dependent outer mitochondrial membrane (OMM)-associated degradation (OMMAD) is critical for mitochondrial and cellular homeostasis. However, the scope and molecular mechanisms of the OMMAD pathways are still not well understood. We report that the OMM-associated E3 ubiquitin ligase MARCH5 controls dynamin-related protein 1 (Drp1)-dependent mitochondrial fission and cell sensitivity to stress-induced apoptosis. MARCH5 knockout selectively inhibited ubiquitination and proteasomal degradation of MiD49, a mitochondrial receptor of Drp1, and consequently led to mitochondrial fragmentation. Mitochondrial fragmentation in MARCH5−/− cells was not associated with inhibition of mitochondrial fusion or bioenergetic defects, supporting the possibility that MARCH5 is a negative regulator of mitochondrial fission. Both MARCH5 re-expression and MiD49 knockout in MARCH5−/− cells reversed mitochondrial fragmentation and reduced sensitivity to stress-induced apoptosis. These findings and data showing MARCH5-dependent degradation of MiD49 upon stress support the possibility that MARCH5 regulation of MiD49 is a novel mechanism controlling mitochondrial fission and, consequently, the cellular response to stress.
Transient assembly of F-actin on the outer mitochondrial membrane contributes to mitochondrial fission
In addition to established membrane remodeling roles in various cellular locations, actin has recently emerged as a participant in mitochondrial fission. However, the underlying mechanisms of its participation remain largely unknown. We report that transient de novo F-actin assembly on the mitochondria occurs upon induction of mitochondrial fission and F-actin accumulates on the mitochondria without forming detectable submitochondrial foci. Impairing mitochondrial division through Drp1 knockout or inhibition prolonged the time of mitochondrial accumulation of F-actin and also led to abnormal mitochondrial accumulation of the actin regulatory factors cortactin, cofilin, and Arp2/3 complexes, suggesting that disassembly of mitochondrial F-actin depends on Drp1 activity. Furthermore, down-regulation of actin regulatory proteins led to elongation of mitochondria, associated with mitochondrial accumulation of Drp1. In addition, depletion of cortactin inhibited Mfn2 down-regulation– or FCCP-induced mitochondrial fragmentation. These data indicate that the dynamic assembly and disassembly of F-actin on the mitochondria participates in Drp1-mediated mitochondrial fission.
Mitochondrial calcium uptake
Calcium (Ca 2+ ) uptake into the mitochondrial matrix is critically important to cellular function. As a regulator of matrix Ca 2+ levels, this flux influences energy production and can initiate cell death. If large, this flux could potentially alter intracellular Ca 2+ ([Ca 2+ ] i ) signals. Despite years of study, fundamental disagreements on the extent and speed of mitochondrial Ca 2+ uptake still exist. Here, we review and quantitatively analyze mitochondrial Ca 2+ uptake fluxes from different tissues and interpret the results with respect to the recently proposed mitochondrial Ca 2+ uniporter (MCU) candidate. This quantitative analysis yields four clear results: ( i ) under physiological conditions, Ca 2+ influx into the mitochondria via the MCU is small relative to other cytosolic Ca 2+ extrusion pathways; ( ii ) single MCU conductance is ∼6–7 pS (105 mM [Ca 2+ ]), and MCU flux appears to be modulated by [Ca 2+ ] i , suggesting Ca 2+ regulation of MCU open probability ( P O ); ( iii ) in the heart, two features are clear: the number of MCU channels per mitochondrion can be calculated, and MCU probability is low under normal conditions; and ( iv ) in skeletal muscle and liver cells, uptake per mitochondrion varies in magnitude but total uptake per cell still appears to be modest. Based on our analysis of available quantitative data, we conclude that although Ca 2+ critically regulates mitochondrial function, the mitochondria do not act as a significant dynamic buffer of cytosolic Ca 2+ under physiological conditions. Nevertheless, with prolonged (superphysiological) elevations of [Ca 2+ ] i , mitochondrial Ca 2+ uptake can increase 10- to 1,000-fold and begin to shape [Ca 2+ ] i dynamics.