Heidi M. McBride

Rab proteins as membrane organizers

Cellular organelles in the exocytic and endocytic pathways have a distinctive spatial distribution and communicate through an elaborate system of vesiculo-tubular transport. Rab proteins and their effectors coordinate consecutive stages of transport, such as vesicle formation, vesicle and organelle motility, and tethering of vesicles to their target compartment. These molecules are highly compartmentalized in organelle membranes, making them excellent candidates for determining transport specificity and organelle identity.

Mitochondria: More Than Just a Powerhouse

Pioneering biochemical studies have long forged the concept that the mitochondria are the energy powerhouse of the cell. These studies, combined with the unique evolutionary origin of the mitochondria, led the way to decades of research focusing on the organelle as an essential, yet independent, functional component of the cell. Recently, however, our conceptual view of this isolated organelle has been profoundly altered with the discovery that mitochondria function within an integrated reticulum that is continually remodeled by both fusion and fission events. The identification of a number of proteins that regulate these activities is beginning to provide mechanistic details of mitochondrial membrane remodeling. However, the broader question remains regarding the underlying purpose of mitochondrial dynamics and the translation of these morphological transitions into altered functional output. One hypothesis has been that mitochondrial respiration and metabolism may be spatially and temporally regulated by the architecture and positioning of the organelle. Recent evidence supports and expands this idea by demonstrating that mitochondria are an integral part of multiple cell signaling cascades. Interestingly, proteins such as GTPases, kinases and phosphatases are involved in bi-directional communication between the mitochondrial reticulum and the rest of the cell. These proteins link mitochondrial function and dynamics to the regulation of metabolism, cell-cycle control, development, antiviral responses and cell death. In this review we will highlight the emerging evidence that provides molecular definition to mitochondria as a central platform in the execution of diverse cellular events.

The PINK1/Parkin pathway regulates mitochondrial morphology

Loss-of-function mutations in the PTEN-induced kinase 1 (PINK1) or parkin genes, which encode a mitochondrially localized serine/threonine kinase and a ubiquitin-protein ligase, respectively, result in recessive familial forms of Parkinsonism. Genetic studies in Drosophila indicate that PINK1 acts upstream of Parkin in a common pathway that influences mitochondrial integrity in a subset of tissues, including flight muscle and dopaminergic neurons. The mechanism by which PINK1 and Parkin influence mitochondrial integrity is currently unknown, although mutations in the PINK1 and parkin genes result in enlarged or swollen mitochondria, suggesting a possible regulatory role for the PINK1/Parkin pathway in mitochondrial morphology. To address this hypothesis, we examined the influence of genetic alterations affecting the machinery that governs mitochondrial morphology on the PINK1 and parkin mutant phenotypes. We report that heterozygous loss-of-function mutations of drp1, which encodes a key mitochondrial fission-promoting component, are largely lethal in a PINK1 or parkin mutant background. Conversely, the flight muscle degeneration and mitochondrial morphological alterations that result from mutations in PINK1 and parkin are strongly suppressed by increased drp1 gene dosage and by heterozygous loss-of-function mutations affecting the mitochondrial fusion-promoting factors OPA1 and Mfn2. Finally, we find that an eye phenotype associated with increased PINK1/Parkin pathway activity is suppressed by perturbations that reduce mitochondrial fission and enhanced by perturbations that reduce mitochondrial fusion. Our studies suggest that the PINK1/Parkin pathway promotes mitochondrial fission and that the loss of mitochondrial and tissue integrity in PINK1 and parkin mutants derives from reduced mitochondrial fission.

Bax/Bak promote sumoylation of DRP1 and its stable association with mitochondria during apoptotic cell death.

Dynamin-related protein 1 (DRP1) plays an important role in mitochondrial fission at steady state and during apoptosis. Using fluorescence recovery after photobleaching, we demonstrate that in healthy cells, yellow fluorescent protein (YFP)–DRP1 recycles between the cytoplasm and mitochondria with a half-time of 50 s. Strikingly, during apoptotic cell death, YFP-DRP1 undergoes a transition from rapid recycling to stable membrane association. The rapid cycling phase that characterizes the early stages of apoptosis is independent of Bax/Bak. However, after Bax recruitment to the mitochondrial membranes but before the loss of mitochondrial membrane potential, YFP-DRP1 becomes locked on the membrane, resulting in undetectable fluorescence recovery. This second phase in DRP1 cycling is dependent on the presence of Bax/Bak but independent of hFis1 and mitochondrial fragmentation. Coincident with Bax activation, we detect a Bax/Bak-dependent stimulation of small ubiquitin-like modifier-1 conjugation to DRP1, a modification that correlates with the stable association of DRP1 with mitochondrial membranes. Altogether, these data demonstrate that the apoptotic machinery regulates the biochemical properties of DRP1 during cell death.

Cargo-Selected Transport from the Mitochondria to Peroxisomes Is Mediated by Vesicular Carriers

Mitochondria and peroxisomes share a number of common biochemical processes, including the beta oxidation of fatty acids and the scavenging of peroxides. Here, we identify a new outer-membrane mitochondria-anchored protein ligase (MAPL) containing a really interesting new gene (RING)-finger domain. Overexpression of MAPL leads to mitochondrial fragmentation, indicating a regulatory function controlling mitochondrial morphology. In addition, confocal- and electron-microscopy studies of MAPL-YFP led to the observation that MAPL is also incorporated within unique, DRP1-independent, 70-100 nm diameter mitochondria-derived vesicles (MDVs). Importantly, vesicles containing MAPL exclude another outer-membrane marker, TOM20, and vesicles containing TOM20 exclude MAPL, indicating that MDVs selectively incorporate their cargo. We further demonstrate that MAPL-containing vesicles fuse with a subset of peroxisomes, marking the first evidence for a direct relationship between these two functionally related organelles. In contrast, a distinct vesicle population labeled with TOM20 does not fuse with peroxisomes, indicating that the incorporation of specific cargo is a primary determinant of MDV fate. These data are the first to identify MAPL, describe and characterize MDVs, and define a new intracellular transport route between mitochondria and peroxisomes.

Sumo1 Conjugates Mitochondrial Substrates and Participates in Mitochondrial Fission

Mitochondrial fission requires the evolutionarily conserved dynamin related protein (DRP1), which is recruited from the cytosol to the mitochondrial outer membrane to coordinate membrane scission. Currently, the mechanism of recruitment and assembly of DRP1 on the mitochondria is unclear. Here, we identify Ubc9 and Sumo1 as specific DRP1-interacting proteins and demonstrate that DRP1 is a Sumo1 substrate. In addition, a surprising number of Sumo1 conjugates were observed in the mitochondrial fractions, suggesting that sumoylation is a common mitochondrial modification. Video microscopy demonstrates that YFP:Sumo1 is often found at the site of mitochondrial fission and remains tightly associated to the tips of fragmented mitochondria. Consistent with this, fluorescence microscopy revealed that a portion of total cytosolic YFP:Sumo1 colocalizes with endogenous mitochondrial DRP1. Finally, transient transfection of Sumo1 dramatically increases the level of mitochondrial fragmentation. Analysis of endogenous DRP1 levels indicates that overexpression of Sumo1 specifically protects DRP1 from degradation, resulting in a more stable, active pool of DRP1, which at least partially accounts for the excess fragmentation. Together, these data are the first to identify a function for Sumo1 on the mitochondria and suggest a novel role for the participation of Sumo1 in mitochondrial fission.

Endoplasmic reticulum BIK initiates DRP1-regulated remodelling of mitochondrial cristae during apoptosis

The endoplasmic reticulum (ER) can elicit proapoptotic signalling that results in transmission of Ca2+ to the mitochondria, which in turn stimulates recruitment of the fission enzyme DRP1 to the surface of the organelle. Here, we show that BH3‐only BIK activates this pathway at the ER in intact cells, resulting in mitochondrial fragmentation but little release of cytochrome c to the cytosol. The BIK‐induced transformations in mitochondria are dynamic in nature and involve DRP1‐dependent remodelling and opening of cristae, where the major stores of cytochrome c reside. This novel function for DRP1 is distinct from its recognized role in regulating mitochondrial fission. Selective permeabilization of the outer membrane with digitonin confirmed that BIK stimulation results in mobilization of intramitochondrial cytochrome c. Of note, BIK can cooperate with a weak BH3‐only protein that targets mitochondria, such as NOXA, to activate BAX by a mechanism that is independent of DRP1 enzyme activity. When expressed together, BIK and NOXA cause rapid release of mobilized cytochrome c and activation of caspases.

MAPL is a new mitochondrial SUMO E3 ligase that regulates mitochondrial fission.

The modification of proteins by the small ubiquitin‐like modifier (SUMO) is known to regulate an increasing array of cellular processes. SUMOylation of the mitochondrial fission GTPase dynamin‐related protein 1 (DRP1) stimulates mitochondrial fission, suggesting that SUMOylation has an important function in mitochondrial dynamics. The conjugation of SUMO to its substrates requires a regulatory SUMO E3 ligase; however, so far, none has been functionally associated with the mitochondria. By using biochemical assays, overexpression and RNA interference experiments, we characterized the mitochondrial‐anchored protein ligase (MAPL) as the first mitochondrial‐anchored SUMO E3 ligase. Furthermore, we show that DRP1 is a substrate for MAPL, providing a direct link between MAPL and the fission machinery. Importantly, the large number of unidentified mitochondrial SUMO targets suggests a global role for SUMOylation in mitochondrial function, placing MAPL as a crucial component in the regulation of multiple conjugation events.

The SUMO protease SENP5 is required to maintain mitochondrial morphology and function

Mitochondria are dynamic organelles that undergo regulated fission and fusion events that are essential to maintain metabolic stability. We previously demonstrated that the mitochondrial fission GTPase DRP1 is a substrate for SUMOylation. To further understand how SUMOylation impacts mitochondrial function, we searched for a SUMO protease that may affect mitochondrial dynamics. We demonstrate that the cytosolic pool of SENP5 catalyzes the cleavage of SUMO1 from a number of mitochondrial substrates. Overexpression of SENP5 rescues SUMO1-induced mitochondrial fragmentation that is partly due to the downregulation of DRP1. By contrast, silencing of SENP5 results in a fragmented and altered morphology. DRP1 was stably mono-SUMOylated in these cells, suggesting that SUMOylation leads to increased DRP1 mediated fission. In addition, the reduction of SENP5 levels resulted in a significant increase in the production of free radicals. Reformation of the mitochondrial tubules by expressing the dominant interfering DRP1 or by RNA silencing of endogenous DRP1 protein rescued both the morphological aberrations and the increased production of ROS induced by downregulation of SENP5. These data demonstrate the importance of SENP5 as a new regulator of SUMO1 proteolysis from mitochondrial targets, impacting mitochondrial morphology and metabolism.

Dissociating the dual roles of apoptosis‐inducing factor in maintaining mitochondrial structure and apoptosis

The mitochondrial protein apoptosis‐inducing factor (AIF) translocates to the nucleus and induces apoptosis. Recent studies, however, have indicated the importance of AIF for survival in mitochondria. In the absence of a means to dissociate these two functions, the precise roles of AIF remain unclear. Here, we dissociate these dual roles using mitochondrially anchored AIF that cannot be released during apoptosis. Forebrain‐specific AIF null (tel. AifΔ) mice have defective cortical development and reduced neuronal survival due to defects in mitochondrial respiration. Mitochondria in AIF deficient neurons are fragmented with aberrant cristae, indicating a novel role of AIF in controlling mitochondrial structure. While tel. AifΔ Apaf1−/− neurons remain sensitive to DNA damage, mitochondrially anchored AIF expression in these cells significantly enhanced survival. AIF mutants that cannot translocate into nucleus failed to induce cell death. These results indicate that the proapoptotic role of AIF can be uncoupled from its physiological function. Cell death induced by AIF is through its proapoptotic activity once it is translocated to the nucleus, not due to the loss of AIF from the mitochondria.