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SHOWCASE ON RESEARCH f F Hungry for Power: Elimination of Mitochondria by Mitophagy Alexander May and Mark Prescott* Department of Biochemistry and Molecular Biology, and ARC Centre of Excellence for Structural and Functional Microbial Genomics, Monash University, Clayton, Victoria 3800 *Corresponding author: [email protected] Introduction It is often said that nothing in life is free, and the role played by the mitochondrion in eukaryotic cells is no exception. Through oxidative phosphorylation, mitochondria provide large amounts of energy to the cell, which has made possible an energy-intensive way of life and, ultimately, the evolution of increasingly complex organisms. The cost, however, is that the production of energy by this unique organelle produces many potentially dangerous by-products, most notably reactive oxygen species (ROS), which can damage a cell’s internal structures, especially as the population of mitochondria accumulate damage and age. The cell’s relationship with its mitochondria must therefore be one of balance, harnessing energy while mitochondrial ATP production is efficient and removing mitochondria once they present a hazard to the eukaryotic cell. The challenges of removing the bulk of an entire organelle, which is beyond the capacity of protein degradation systems such as the proteasome, are overcome by utilising the autophagic machinery of the cell. In broad terms, this involves the isolation of cargo (in this case mitochondria) into a double-membrane structure known as the autophagosome and its delivery, via membrane fusion, to the lysosome (in mammalian cells) or the vacuole (in yeast) for degradation (Fig. 1, reviewed in (1)). This autophagy of mitochondria, termed mitophagy (2), is a controlled process that achieves selective removal of the organelle in response to intracellular cues and extracellular stimuli. In addition to the underlying quality control function, mitophagy is employed to adjust mitochondrial mass during changes in the availability of nutrients and during stationary phase as yeast cells adapt to their environment, whereas the process is observed during development and immune cell Fig. 1. A simplified illustration of how autophagy, specifically macroautophagy, occurs in yeast. Initiation of autophagy is followed by a membrane expansion event that ultimately encloses components destined for degradation in the autophagosome. The autophagosome then traffics to and fuses with the vacuole or lysosome, releasing its contents into the lumen for degradation by hydrolases. Finally, degraded components are returned to the cytoplasm for reuse. Microautophagy (not shown) is also an important autophagic route in yeast. Page 4 AUSTRALIAN BIOCHEMIST Vol 42 No 2 August 2011 Elimination of Mitochondria by Mitophagy SHOWCASE ON RESEARCH Fig. 2. Mitophagy in yeast. According to one current model, mitochondrial stress (!) is sensed by Atg33p, which relays the signal on to Atg32p, and in turn recruits core Atg proteins. Atg11p has been implicated in all types of selective autophagy in yeast and most likely plays a scaffolding role in the interaction between Atg32p and Atg8p. Stressed mitochondria can then be enclosed in an autophagosome and delivered to the vacuole for removal and recycling. maturation in mammalian cells. Although conceptually simple, the various roles of mitophagy suggest mechanistic and regulatory complexity, offering a fascinating insight into the cell’s Faustian pact with the mitochondrion. Mitophagy in Yeast Much of our knowledge of mitophagy has been obtained through experiments examining the budding yeast Saccharomyces cerevisiae, the key model organism of the field. In addition to numerous ‘core’ ATG (autophagy) genes, which are involved in all autophagic processes, mitophagy in yeast relies on a number of additional genes, the functions of many of which are yet to be elucidated. Genetic screens by two separate groups have identified two genes, ATG32 and ATG33, which are specific to mitophagy (3,4). The precise function of their encoded proteins is not fully understood; the outer membrane (OM) protein Atg32p is thought to play a central role in bringing a mitochondrion selected for degradation into contact with the core autophagic machinery, whereas Atg33p, also on the OM, is implicated in the sensing of stress that triggers mitochondrial degradation (Fig. 2). Once mitophagy is triggered, the selected mitochondrion is delivered to the core autophagic machinery through an interaction of Atg32p with Atg8p, the protein behind the biogenesis and expansion of the autophagosome and the homolog of mammalian LC3. The interaction between Atg32p and Atg8p is facilitated by an Atg8 interacting Fig. 3. Mitophagy in mammalian cells. A. In the NIX-dependent pathway, associated with reticulocyte maturation, LC3 is recruited following the recognition of mitochondrial stress (!), which induces autophagosome formation. B. Mitochondrial stress can also be recognised by PINK1, which recruits Parkin. Parkin then ubiquitinates yet unknown factors (?) that possibly initiate mitophagy. Both pathways result in delivery to the lysosome and degradation. Vol 42 No 2 August 2011 AUSTRALIAN BIOCHEMIST Page 5 Elimination of Mitochondria by Mitophagy SHOWCASE ON RESEARCH motif (AIM), which is crucial to efficient mitophagy. The adaptor protein Atg11p, which is essential for all selective forms of autophagy identified to date, is also indispensible in this interaction. Additionally, a number of proteins originally identified in targeted studies by their function in other processes have also been implicated in mitophagy. These include Uth1p (a SUN family protein involved in ageing and cell wall biogenesis (5)), Aup1p (involved in the retrograde signalling pathway between the mitochondrion and nucleus (6)) and Mdm38p (a mitochondrial inner membrane protein associated with K+/H+ exchange (7)). The diversity of proteins involved in mitophagy reinforces the point that the degradation of mitochondria is an important and complex cellular activity, enmeshed as a basic process in the cellular milieu. However, our inability to describe this process in any detail beyond a basic model also suggests that many proteins involved in mitophagy are yet to be uncovered. Clearly, much work remains to be undertaken in the field. Mitophagy in Mammals The results of research on mammalian cells have proven to be equally intriguing, and further research has been spurred on mainly by the association of perturbed mitochondrial turnover with several neurodegenerative disorders, including Parkinson’s disease and Alzheimer’s disease (8). At the cellular level, mitochondrial integrity appears to be maintained through mitochondrial dynamics, with poorly performing mitochondria – as measured by a loss of membrane potential – being isolated by fission and prepared for degradation (9). Although similarities in general autophagic mechanisms are apparent when considering mammalian and yeast cells, the mechanism behind mitophagy at the level of the organelle appears to differ in mammalian cells from that of yeast, and mammalian homologues to Atg32p and Atg33p are yet to be described. However, proteins specific to mitophagy in mammalian cells have been identified and offer an insight into how mitochondria are selected for removal. In mammalian cells, mitophagy is implicated in a range of cellular functions in addition to that of the quality control of mitochondria. An example is the maturation of reticulocytes, a process requiring the removal of the cell’s entire population of mitochondria as the cells develop into erythrocytes. This process has been found to be mediated by a mitochondrial OM protein, NIP3-like protein X (NIX) (10) (Fig. 3A). Like the yeast protein Atg32p, the Bcl-2 family protein NIX contains an AIM, which is responsible for interaction with the mammalian homologue of Atg8, LC3. This interaction brings mitochondria into contact with the autophagic machinery, allowing mitophagy to occur. In reticulocytes, NIX expression is upregulated immediately before the elimination of the cell’s mitochondria, and deletion of the gene is observed to block mitochondrial elimination. At the same time, overexpression of another Bcl-2 family protein, Bnip3, has also been shown to result in increased mitophagy (11). While Bcl-2 family proteins are implicated in apoptosis, it seems to be their ability to alter mitochondrial membrane potential – a crucial factor in initiating mitophagy – that is behind autophagic degradation. Fig. 4. Schematic diagram of Rosella. The biosensor incorporates a red fluorescent protein (DsRed T3) and pH-sensitive green fluorescent protein (SEP) joined by a short linker region (grey region). A targeting sequence occupies the N-terminus. Excitation (Ex) and emission (Em) wavelengths are indicated above. Fig. 5. Output of the Rosella assay. The pHsensitive green fluorescent protein of mitochondriatargeted Rosella fluoresces only at cytosolic pH, whereas the red-emitting fluorescent protein fluoresces at both cytosolic and vacuolar pH. In wild-type cells (shown here) cultured in nutrient-rich conditions, red and green emission is absent from the vacuole (A). However, upon removal of nitrogen from the medium (starvation) vacuolar delivery of mitochondria (mitophagy), a characteristic red vacuolar fluorescence is observed (B, white arrows). Page 6 AUSTRALIAN BIOCHEMIST Vol 42 No 2 August 2011 Elimination of Mitochondria by Mitophagy SHOWCASE ON RESEARCH In addition to the NIX- and Bnip3-mediated induction of mitophagy, the literature suggests the involvement of other proteins in the priming of mitochondria for elimination (Fig. 3B). Two proteins, PINK1 and PARKIN, have received particular attention due to their association with neurodegenerative disorders. In particular, PARKIN loss-of-function mutations are the most prevalent genetic mutation associated with early-onset Parkinson’s disease (12). From available data, it is hypothesised that the mitochondrial OM-localised Ser/Thr kinase PINK1 plays a role in the detection of mitochondrial stress by an unknown mechanism. PINK1 then communicates this stress by binding PARKIN, a cytosolic ubiquitin ligase, which subsequently ubiquitinates target proteins, bringing about mitophagy (13). The targets of ubiquitination are not clearly understood, but it is likely that they promote the fission of stressed organelles from the greater mitochondrial network for degradation, as perturbation of either of these genes results in swollen mitochondrial morphology in cells. Future Research While the cooperation of such sensing and induction mechanisms offers an attractive model of how mitophagy occurs in mammalian cells, more work is essential to describe the process at a level of detail useful for clinically oriented research. For example, very little is known about the regulation of mitophagy in both yeast and mammalian cells. As with many autophagic processes, it is reasonable to assume that, due to the fundamental role mitophagy plays in cellular homeostasis, much more remains to be learnt about the mechanisms of this process and its role in the maintenance of homeostasis in cells. With the rapid pace of research in the field, the coming years will see the significance of this process reflected in important advances in understanding of the unique relationship between eukaryotic cells and the mitochondrion. At the Monash laboratory, we investigate the mechanisms and regulation of mitophagy by interrogating a number of complementary yeast strain collections using a fluorescence assay based on a novel biosensor known as Rosella (Fig. 4). Rosella incorporates two fluorescent proteins, one red (pHstable) and the other green (pH-sensitive), which are joined by a linker region (14,15). In our studies of mitophagy, Rosella is targeted to the mitochondrial matrix. Green fluorescence emission is strong at pH > 7.0, but is quenched at vacuolar pH (< 6.0), while red fluorescence emission remains strong in the pH range 4 to 11. Accordingly, delivery of Rosella-tagged mitochondria to the acidic lumen of the vacuole is accompanied by the quenching of green but not red fluorescence, as demonstrated by red vacuolar fluorescence in the merged image (Fig. 5B). By scoring the proportion of cells undergoing mitophagy at selected time points under different conditions, and in different strains, an indication of the contribution and importance of individual genes in mitophagy can be obtained. Utilising this approach, we have uncovered the involvement of a number of genes including a previously uncharacterised open reading frame, OTP1, which appears to take part in an early phase of mitophagy, possibly in concert with UTH1, as well as a variety of other genes that appear to play a role in the process. With further research, we hope to establish how OTP1 together with other mitophagy genes contribute to the simultaneously complex and elegant means of mitochondrial homeostasis that is mitophagy. References 1. Xie, Z., and Klionsky, D.J. (2007) Nature Cell Biol. 9, 11021109 2. Lemasters, J.J. (2005) Rejuven. Res. 8, 3-5 3. Kanki, T., Wang, K., Baba, M., Bartholomew, C.R., Lynch-Day, M.A., et al. (2009) Mol. Biol. Cell 20, 47304738 4. 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(2010) PLoS Biol. 8, e1000298 14.Rosado, C.J., Mijaljica, D., Hatzinisiriou, I., Prescott, M., and Devenish, R.J. (2008) Autophagy 4, 205-213 15.Mijaljica, D., Prescott, M., and Devenish, R.J. (2011) J. Vis. Exp. (in press) f Vol 42 No 2 August 2011 F AUSTRALIAN BIOCHEMIST Page 7