- Email: [email protected]
Mitochondrial Proteins Moonlighting in the Nucleus
TIBS 1190 No. of Pages 8
Opinion
Mitochondrial Proteins Moonlighting in the Nucleus Richard M. Monaghan1 and Alan J. Whitmarsh1,* Mitochondria function as cellular energy generators, producing the fuel required to drive biological processes. The response of cells to mitochondrial activity or dysfunction regulates their survival, growth, proliferation, and differentiation. Several proteins that contain mitochondrial-targeting sequences (MTS) also reside in the nucleus and there is increasing evidence that the nuclear translocation of mitochondrial proteins represents a novel pathway by which mitochondria signal their status to the cell. Here, we discuss the different mechanisms that control the dual mitochondrial and nuclear localisation of proteins and propose that these nuclear moonlighters represent a widespread regulatory circuit to maintain mitochondrial homeostasis. Mitochondrial Communication with the Nucleus Mitochondria host many metabolic processes, including the generation of cellular energy in the form of ATP by oxidative phosphorylation (see Glossary). Although they contain their own genome, most mitochondrial proteins are encoded in the nucleus. It is essential for nuclei to sense changes in mitochondrial metabolism and mount an appropriate response to restore homeostasis [1–3]. Disrupted communication between mitochondria and nuclei is implicated in metabolic diseases, cancer, neurodegeneration, and other ageing processes. The role of mitochondria in apoptosis is well characterised [4], but under nonapoptotic conditions, retrograde signalling pathways from mitochondria to nuclei are triggered by changes in metabolite levels or altered proteostasis [2,3,5]. Commonly, metabolites, including reactive oxygen species (ROS), act as second messengers to regulate cytoplasmic pathways [2,3,5]. However, a new paradigm is emerging whereby proteins that harbour an MTS can localise to nuclei and, in some cases, act as direct signals from mitochondria to regulate nuclear events. We propose that, by acting as signalling intermediaries between mitochondria and nuclei, these proteins promote a rapid response to changes in mitochondrial function and may directly link metabolic activity to genome integrity and gene expression.
Trends An increasing number of mitochondrial proteins have been reported to reside in the nucleus and act as mediators of direct mitochondria-to-nucleus communication. These proteins may perform similar functions in both compartments or have distinct activities. Mitochondrial protein import is responsive to mitochondrial activity and this represents a key regulatory point to determine the balance between mitochondrial and nuclear localisation. Active mitochondrial enzyme complexes may translocate from the mitochondrial matrix to nuclei. Metabolic enzymes can associate with chromatin and regulate gene expression.
Direct Mitochondria-to-Nucleus Signalling Conceptually, the translocation of mitochondrial proteins to nuclei represents the simplest and most direct means of retrograde communication between the two organelles. These proteins can be responsive to a variety of stimuli and may perform a similar function in both compartments or have distinct mitochondrial and nuclear roles. Below, we discuss some of the mechanisms involved. Nuclear Redirection of Transcription Factors in Response to Disrupted Mitochondrial Function Transcription factors can be targeted to mitochondria yet primed to be redirected to the nucleus in response to mitochondrial stress. For example, mammalian nuclear factor erythroid 2-related factor 2 (NRF2), which lacks an MTS, is sequestered in a complex with Kelch-like ECHassociated protein 1 (KEAP1) and the MTS-containing phosphatase phosphoglycerate mutase
Trends in Biochemical Sciences, Month Year, Vol. xx, No. yy
1 Faculty of Life Sciences, University of Manchester, Michael Smith Building, Oxford Road, Manchester, M13 9PT, UK
*Correspondence: [email protected] (A.J. Whitmarsh).
http://dx.doi.org/10.1016/j.tibs.2015.10.003 © 2015 Elsevier Ltd. All rights reserved.
1
TIBS 1190 No. of Pages 8
Nucleus
Glossary
Mitochondrion NRF2
PGAM5
MTS
KEAP1
Oxidave stress
ATFS-1 Disrupted proteostasis
MTS
ATFS-1
MTS
NRF2 Anoxidant response
ATFS-1
UPRmt
Histone acetylaon
CLK-1
MTS
ROS barometer
mtDNA
MTS
CLK-1
OXPHOS
CLK-1
Acetyl-CoA
Acetyl-CoA Growth signals PDC
ETS inhibion PDC proteins
MTS
PDC
Figure 1. Mitochondrial Proteins Directly Signal to the Nucleus to Regulate Gene Expression. In mammalian cells, the transcription factor NRF2 associates with the outer mitochondrial membrane as part of a complex with KEAP1 and PGAM5, but dissociates from the complex upon oxidative stress and translocates to the nucleus, where it targets the promoters of genes that contribute to antioxidant defences [6–8]. The Caenorhabditis elegans transcription factor ATFS-1 is imported into the mitochondrial matrix and degraded by proteolysis [9], while CLK-1 and components of the PDC are imported into mitochondria and their N-terminal MTSs cleaved [16,20]. CLK-1 is required for the synthesis of ubiquinone and PDC generates acetyl-CoA, both of which contribute to oxidative phosphorylation (OXPHOS) [15,22]. ATFS-1 and CLK-1 are redirected from mitochondria to nuclei in response to disrupted proteostasis and [7_TD$IF]physiological ROS [8_TD$IF]production, respectively [9,16]. ATFS-1 mediates the UPRmt and the expression of mitochondrial-encoded genes in C. elegans [9,12], while CLK-1 acts to limit stress responses in both C. elegans and human cells [16]. PDC is proposed to translocate from mitochondria to nuclei as an intact complex in human cells in response to growth signals or impaired oxidative phosphorylation. Similar to its mitochondrial activity, nuclear PDC generates acetyl-CoA and this is used as a co-factor for histone acetylation [20]. Abbreviations: ATFS-1, activating transcription factor associated with stress 1; CLK-1, CLOCK-1; CoA, coenzyme A; ETS, electron transport system; KEAP1, Kelch-like ECH-associated protein 1; MTS, mitochondrial-targeting sequence; NRF2, nuclear factor erythroid 2-related factor 2; OXPHOS, oxidative phosphorylation; PDC, pyruvate dehydrogenase complex; PGAM5, phosphoglycerate mutase 5; ROS, reactive oxygen species.
5 (PGAM5) at the outer mitochondrial membrane [6,7]. Upon mitochondrial stress, NRF2 dissociates from the complex and translocates to the nucleus to activate its target genes [6] (Figure 1). This close proximity with the primary site of cellular ROS production allows NRF2 to rapidly mobilise antioxidant defences when mitochondrial oxidative stress occurs [8]. In contrast to NRF2, the nuclear localisation of the Caenorhabditis elegans transcription factor Activating Transcription Factor associated with Stress 1 (ATFS-1) is regulated by the efficiency of its mitochondrial import [9]. ATFS-1 is a component of the mitochondrial unfolded protein response (UPRmt[14_TD$IF]), which is triggered by disrupted proteostasis [10,11]. Normally, ATFS-1 is imported into mitochondria, where it undergoes proteolytic degradation; however, upon UPRmt induction, it is stabilised within the mitochondria and interacts with the mitochondrial genome to limit the accumulation of mitochondrially encoded mRNAs [9,12]. Concurrently, mitochondrial import of ATFS-1 is impaired and a pool is redirected to the nucleus, where it regulates genes encoding proteins required for maintaining proteostasis and oxidative phosphorylation [9,12] (Figure 1). ATFS-1 is able to localise to both organelles because it contains an MTS and a nuclear localisation signal; it is the coordinated mitochondrial stabilisation and nuclear localisation of
2
Trends in Biochemical Sciences, Month Year, Vol. xx, No. yy
Echoforms: identical forms of a protein at different subcellular locations. Eclipsed distribution: the uneven distribution of a protein between two cellular compartments. Endosymbiosis: the theory that organelles, such as mitochondria, originated as a symbiosis between two distinct prokaryotic cells. Homeostasis: the maintenance of a constant internal environment in cells or organisms to maintain functioning by compensating for changing conditions. Mitochondrial targeting sequence (MTS): a short amino acid sequence usually found near the N terminus of nuclear-encoded mitochondrial proteins that can interact with the import machinery located within the inner and outer mitochondrial membranes. Mitochondrial unfolded protein response (UPRmt): a mitochondriato-nucleus signalling pathway that responds to mitochondrial stress, particularly the accumulation of misfolded or unfolded proteins in mitochondria or an imbalance between mitochondrial and nuclear encoded proteins. Mitophagy: the selective degradation of damaged mitochondria by autophagy. Moonlighting protein: a protein with more than one function. Oxidative phosphorylation (OXPHOS): the process by which electrons are transferred to oxygen by the electron transport chain located at the mitochondrial inner membrane to produce energy in the form of ATP. Proteostasis: the coordinated regulation of proteins including their translation, folding, movement, and degradation. Cells try to maintain proteostasis, particularly in response to stress. Reactive oxygen species (ROS): chemically reactive molecules that contain oxygen. Mitochondria are the major site of ROS generation in cells because ROS are a by-product of oxidative phosphorylation. Retrograde signalling: in the context of this article, this refers to signalling from mitochondria to the nucleus. Signalling from the nucleus to other organelles is termed anterograde signalling.
TIBS 1190 No. of Pages 8
ATFS-1 that allows it to act as a sensor of mitochondrial stress and promote the recovery of oxidative phosphorylation [9,12]. The UPRmt is a conserved retrograde pathway in eukaryotes [10,11], but is relatively poorly characterised in mammals. The transcription factors C/EBP homologous protein (CHOP) and CCAAT-enhancer binding protein b (C/EBPb) have been proposed to coordinate mammalian UPRmt target gene expression, but they are not direct signalling intermediaries between mitochondria and nuclei [13,14]. Therefore, it will be interesting to determine whether functional homologues of ATFS-1 exist in mammalian cells. Maintaining Mitochondrial Homeostasis It is important that the stress-response programmes, such as those mediated by NRF2 and ATFS-1, are carefully monitored. The mitochondrial enzyme CLOCK-1 (CLK-1; also called COQ7) may have a key role in this. CLK-1 catalyses a step in the biosynthesis of ubiquinone, a component of the electron transport system [15], and was recently shown to reside in nuclei and regulate gene expression in both C. elegans and human cells [16]. CLK-1 is present in the nucleus in the absence of mitochondrial stress, but its localisation is dependent on physiological ROS production [16]. Importantly, nuclear CLK-1 suppresses gene expression mediated by ROS-induced stress pathways and the UPRmt, and so it may function to limit the activation of a major stress response until a particular threshold of mitochondrial dysfunction is reached [16]. In this way, the nuclear translocation of CLK-1 acts as a ROS barometer to maintain mitochondrial homeostasis (Figure 1).
Retrotranslocation: in the context of this article, the movement of a protein out of mitochondria contrary to its normal route of import and processing. Second messengers: intracellular signalling molecules released within cells to initiate signal transduction cascades. Tricarboxylic acid (TCA) cycle: a metabolic pathway found in mitochondria that releases electrons to the electron transport chain for oxidative phosphorylation. It also provides the building blocks for the biosynthesis of amino acids, fatty acids, and other biological molecules.
The exact mechanism by which CLK-1 and ATFS-1 are redirected to the nucleus remains unclear. The nuclear forms retain their MTS, indicating that they have not been imported into the mitochondrial matrix (where their MTS would be cleaved by proteases [17]). However, this would not exclude them being initially targeted to the outer mitochondrial membrane or translocated into the intermembrane space before their nuclear translocation. The mechanism by which nuclear CLK-1 regulates gene expression is also not known. CLK-1 is an iron-binding monooxygenase that has hydroxylase activity [18], and it will be important to ascertain whether this activity is required for its nuclear role. If so, given that CLK-1 associates with chromatin [16], it is possible that it could directly modify DNA or histones. While this would be a novel role for a monooxygenase, it is noteworthy that iron-binding dioxygenases have been shown to regulate gene expression through the demethylation of cytosine bases and histones [19]. Double Duty for a Mitochondrial Matrix Complex In contrast to ATFS-1 and CLK-1, the human pyruvate dehydrogenase complex (PDC) appears to translocate directly from the mitochondrial matrix to the nucleus [20]. This is supported by the observation that nuclear PDC components lack their MTS, implying prior processing in the mitochondrial matrix [20,21]. PDC links cytoplasmic glycolytic metabolism to mitochondrial oxidative phosphorylation by converting pyruvate to the tricarboxylic acid (TCA) cycle substrate acetyl co-enzyme A (-CoA) [22]. It performs the same enzymatic function in the nucleus, providing a pool of acetyl-CoA that is used as a cofactor for histone acetylation [20]. Nuclear PDC levels are cell cycle dependent and increase in response to growth signals or the inhibition of oxidative phosphorylation [20]. Therefore, PDC coordinates mitochondrial metabolism with nuclear gene expression to regulate cell growth (Figure 1). The proposal that PDC translocates from the mitochondrial matrix to the nucleus as an intact complex is intriguing. To do this, PDC must traverse both mitochondrial membranes before moving to the nucleus. The nuclear import of large complexes is well characterised [23], but their mitochondrial export is not. Mitochondrial vesicles have recently been identified as a novel mechanism to selectively degrade discrete regions of mitochondrial membranes and associated proteins [24]. PDC components have been detected in these [25], so it is inviting to speculate that a nondegradatory vesicular pathway exists in which proteins destined for locations
Trends in Biochemical Sciences, Month Year, Vol. xx, No. yy
3
TIBS 1190 No. of Pages 8
secondary to the mitochondria, for example the nucleus, are redirected. Another possibility is that PDC components retrotranslocate back out of the mitochondria following cleavage of their N-terminal MTS and form a complex in the cytosol that translocates to the nucleus. Alternatively, leakage or breakdown of the mitochondrial membrane could occur, even under nonapoptotic conditions, allowing proteins to exit from the mitochondrial matrix.
Dual Targeting to Mitochondria and Nuclei The proteins discussed so far directly link mitochondrial activity to nuclear events. However, for other MTS-containing proteins that are targeted to both compartments, it is less clear whether the two protein pools communicate with each other or act separately. For example, the yeast enzyme Arg5,6 is required for arginine biosynthesis in mitochondria and regulates nuclear gene expression, but whether it integrates these two processes is not known [26]. In some cases, these proteins may constitute an anterograde pathway from the nucleus to mitochondria. Alternatively, regulating the relative amounts of the protein in each compartment may indirectly affect the function of the other organelle. Dual Targeting of Fumarase One of the best-characterised dual-targeted proteins is the enzyme fumarase, which participates in the TCA cycle in mitochondria and regulates the response to DNA damage in the nucleus [27] (Figure 2). The two pools of fumarase have been coined [3_TD$IF]echoforms[4_TD$IF], a term applicable to identical forms of a protein that are found at different cellular locations [28]. The respective rates of protein translation and mitochondrial import control the mitochondrial
Nucleus
Mitochondrion
Telomere maintenance
TIN2
MTS
MTS
TPP1 TIN2
TIN2
TERT
MTS
MTS
TERT
MTS
ROS TERT
mtDNA
RECQL4
RECQL4
p32
Fumarase
Fumarase
TCA cycle
MTS
Fumarase
Fumarase
MTS
RECQL4
MTS
DNA damage response
MTS
ROS
Figure 2. Dual-Targeted Proteins Maintain Nuclear and Mitochondrial Genome Integrity in Response to Stress. Several regulators of nuclear genome stability contain an MTS and are targeted to mitochondria. In mammalian cells, mitochondrial TERT and RECQL4 levels are enhanced by increased ROS, while the balance of nuclear and mitochondrial localisation of both TIN2 and RECQL4 can be regulated by their binding partners, TPP1 and p32, respectively [32–36]. In mitochondria, TERT and RECQL4 help maintain mitochondrial genome integrity and copy number, and protect from ROS stress. TIN2 regulates mitochondrial morphology and promotes ROS production [32]. The TCA cycle enzyme fumarase participates in the nuclear DNA damage response and its localisation in yeast is, in part, controlled by the comparative rates of its mitochondrial import and protein translation [27,29]. Abbreviations: mtDNA, mitochondrial DNA; MTS, mitochondrial-targeting sequence; RECQL4, RecQ helicase-like 4; ROS, reactive oxygen species; TCA, tricarboxylic acid; TERT, telomerase reverse transcriptase; TIN2, TERF1-interacting protein 2; TPP1, [9_TD$IF]TIN2 [10_TD$IF]interacting protein 1.
4
Trends in Biochemical Sciences, Month Year, Vol. xx, No. yy
TIBS 1190 No. of Pages 8
localisation of fumarase in yeast [29]. Fumarase is imported co-translationally; if import slows, it starts to fold in the cytoplasm, thus blocking its further import and resulting in its retrotranslocation back into the cytoplasm [29]. In contrast to ATFS-1 and CLK-1, the N-terminal MTS of fumarase reaches the mitochondrial matrix and is cleaved before retrotranslocation, so both cytosolic and mitochondrial fumarase are the processed forms of the enzyme [29]. In response to DNA double-strand breaks, fumarase is recruited from the cytosol to the nucleus, where it produces fumarate, which contributes to regulating the DNA damage response in both yeast and human cells [27]. Recent evidence suggests that nuclear fumarate promotes DNA repair by inhibiting histone demethylation [30]. Interestingly, the mammalian homologue of fumarase appears to be regulated differently; distinct translation products that are produced from a single gene are differentially localised [29]. Regulating Nuclear and Mitochondrial Genome Stability There are other proteins that are required for maintaining mammalian genome integrity, contain an N-terminal MTS, and display dual mitochondrial and nuclear localisation. These include the telomerase component telomerase reverse transcriptase (TERT), which prevents telomere shortening in the nucleus; the telomeric protein TERF1-interacting protein 2 (TIN2); and the DNA helicase RecQ helicase-like 4 (RECQL4) [31–35] (Figure 2). In mitochondria, TERT protects the mitochondrial genome from oxidative stress [36–39], TIN2 is proposed to regulate oxidative phosphorylation and mitochondrial morphology [32], and RECQL4 controls mitochondrial DNA copy number and integrity [33–35]. The N terminus of TIN2 is processed in the mitochondria, but it is unclear whether this is the case for TERT or RECQL4. The nuclear and mitochondrial pools of these proteins appear to act separately, but the balance between them can be regulated. For example, TERT is predominantly nuclear, but oxidative stress can shift it to be predominantly mitochondrial, indicative of anterograde signalling from nuclei to mitochondria [36]. This suggests that protecting the mitochondrial genome is prioritised over the nuclear genome under these conditions. It is likely that redirecting resources to protect the mitochondrial genome is a survival strategy, because mitochondrial dysfunction can lead to increased local levels of ROS that could damage the nearby mitochondrial DNA.
Advantages of Dual Targeting and Direct Mitochondrial to Nuclear Signalling The targeting of the same protein to different cellular locations appears to be a widespread phenomenon that operates alongside other mechanisms, including alternative splicing or alternative translation, to limit the requirement for gene duplication. Combined with the increasing number of proteins that have been identified as having more than one function [28,40,41], this suggests that there is evolutionary pressure for proteins to acquire additional cellular roles. An advantage of this would be the efficient management of resources. It is evident that many dual-targeted proteins have an eclipsed distribution, meaning that they predominantly localise and function in one compartment, with only a small pool required in the second compartment [42]. Depending on need, the distribution of the protein between compartments may be rapidly modulated and, in some cases, the translocation of the protein between compartments acts as a signal that allows functional integration. For those proteins that translocate from mitochondria to the nucleus, what are the advantages of this direct rerouting compared with signalling through cytosolic pathways? Perhaps by associating with the outer membrane or being integral mitochondrial proteins, they can respond to changes in metabolic activity that secondary messenger levels in the cytoplasm are unable to communicate. Indeed, some responses may only be possible from within mitochondria; for example, [15_TD$IF]in reaction to changes at the inner membrane where oxidative phosphorylation occurs. Of note, the inhibition of mitochondrial protein import may be a common mechanism by which mitochondrial activity is monitored by cells. For example, the impaired mitochondrial import of ATFS-1 and CLK-1 is sufficient to redirect them to the nucleus [9,16]. Also, the selective
Trends in Biochemical Sciences, Month Year, Vol. xx, No. yy
5
TIBS 1190 No. of Pages 8
degradation of mitochondria (mitophagy) and a newly characterised stress pathway in yeast, activated by cytosolic accumulation of toxic mitochondrial precursor proteins, are both mediated by changes to mitochondrial protein import [43–45]. Therefore, it is plausible that a range of mitochondrial states can be communicated by the ability of different proteins to be imported. The import of mitochondrial proteins assists in their folding and integration into complexes [17], and so mitochondrial import may be required as a preceding step to form active proteins before their translocation to the nucleus. This could be the case for PDC, which is proposed to translocate as an active complex to nuclei from the mitochondrial matrix [20]. This would be somewhat analogous to the well-characterised endoplasmic reticulum pathway of protein folding and maturation, and the subsequent transport of proteins to distinct cellular localisations [46]. Bearing in mind that mitochondria originated as a result of endosymbiosis [47], it is conceivable that appropriate conditions for particular proteins to fold and form complexes may uniquely exist within mitochondria and that no alternate cellular environment evolved to fulfil a similar role. Thus, if an extramitochondrial function arose later in evolution, this could only be achieved by translocation from mitochondria following import and correct folding. One consequence of direct signalling from individual mitochondrial sites is that it permits a localised stress to elicit a retrograde response. In such circumstances, a global change in nuclear gene expression would not necessarily be warranted. Therefore, controlling the balance of the activities of different mitochondria-to-nuclear translocation pathways is important. If most mitochondria are functioning normally, this would maintain a pool of nuclear CLK-1 that could dampen retrograde stress responses mediated by NRF2 or ATFS-1 and triggered by a few dysfunctional mitochondria. These mitochondria could be pushed towards mitophagy and degradation [43], rather than mounting a protective programme of gene expression. In this way, the integration of antagonistic pathways that are sensitive to various levels of mitochondrial stress would dictate the response mounted to maintain mitochondrial and cellular homeostasis. A further consequence of[5_TD$IF] translocation[6_TD$IF] away from mitochondria to the nucleus would be to decrease the mitochondrial pool[16_TD$IF] of proteins. Relatively small reductions in the levels of the mitochondrial proteins may not appear relevant for abundant metabolic proteins, such as CLK-1 and PDC, but at the level of an individual mitochondrion they could be. Indeed, autoregulatory loops may operate, whereby redirection to the nucleus decreases the mitochondrial protein pool and directly alters aspects of mitochondrial function. This could lead to inhibition of nuclear localisation and/or promote mitochondrial import of the protein, thereby re-establishing the mitochondrial function. Such a mechanism could operate in tandem with the global response mounted in the nucleus that may reverse the original signal. The integration of multiple regulatory strands would act as a sensitive rheostat of mitochondrial fitness.
Concluding Remarks Considering the importance of maintaining mitochondrial integrity and optimal metabolism for cellular fitness, it is unsurprising that many signalling routes between mitochondria and the nucleus have evolved. The recent studies uncovering direct signalling intermediaries that functionally link these compartments has shifted perceptions of how mitochondria signal (Table 1). Clearly, further investigation into the distinct mechanisms involved is warranted. Importantly, several hundred proteins have been reported to display both mitochondrial and nuclear localisation [48]. A number of these harbour an MTS, but their nuclear roles remain largely uncharacterised (see Outstanding Questions). However, it points to the nuclear moonlighting of mitochondrial proteins being part of an extensive signalling network within cells. This has implications for interpreting studies of proteins previously assumed to be exclusively mitochondrial, because their nuclear roles may contribute to reported functions and associated phenotypes. This is true for PDC and CLK-1, where their nuclear roles have now been shown to
6
Trends in Biochemical Sciences, Month Year, Vol. xx, No. yy
Outstanding Questions What is the full extent of mitochondrial proteins moonlighting in the nucleus? Many proteins have been reported to localise to both compartments and several contain an MTS. It will be important to characterise their nuclear functions. Can the functions and associated phenotypes of mitochondrial proteins, previously presumed to reflect their mitochondrial role, be explained by their newly discovered nuclear roles? How are MTS-containing mitochondrial proteins, such as CLK-1 and ATFS-1, redirected to nuclei? The regulation of mitochondrial import efficiency is clearly important, but the molecular mechanisms involved need to be resolved. How do mitochondrial enzyme complexes, such as PDC, translocate to the nucleus? There is currently no mechanism to explain how a large active enzyme complex can move from the mitochondrial matrix to the nucleus. How do metabolic enzymes regulate gene transcription? CLK-1 and Arg5,6 do not have obvious DNA binding or transcription activation and/or repression domains, so how they alter transcription is unclear. Do they associate with specific transcription factors or modify chromatin? Is their enzymatic activity required?
TIBS 1190 No. of Pages 8
Table 1. MTS-Containing Proteins with Dual Mitochondrial and Nuclear Functions Protein
Species
Mitochondrial Function
Nuclear Function
Interaction between Two Organelles
ATFS-1
Caenorhabditis elegans
Limits mitochondrial mRNA production
Mediates UPRmt[1_TD$IF] by direct regulation of gene expression
Disrupted mitochondrial proteostasis stabilises mitochondrial ATFS-1 and directs pool of protein to nucleus
CLK-1
C. elegans and human
Ubiquinone biosynthesis
Modulates mitochondrial stress responses through regulating gene expression
Localises predominantly to mitochondria with a small amount redirected to nucleus in response to physiological ROS production
PDC
Human
Acetyl-CoA production for the TCA cycle
Acetyl-CoA production for histone acetylation
Translocates from mitochondria to nuclei in response to growth signals or impaired oxidative phosphorylation
Fumarase
Yeast
Fumarate production in TCA cycle
Fumarate production for DNA damage response
Cleaved during mitochondrial import with the nuclear form retrotranslocating from mitochondria
TERT
Human
Protects from telomere shortening
Protects mtDNA from oxidative damage
Oxidative stress increases amount of mitochondrial TERT
TIN2
Human
Regulation of telomeres
Regulates oxidative phosphorylation
Balance between nuclear and mitochondrial localisation regulated by interaction with [9_TD$IF]TIN2 [1_TD$IF]interacting protein 1 [12_TD$IF](TPP1[13_TD$IF])
RECQL4
Human
Maintaining genome stability
Controls mtDNA copy number and integrity
Oxidative stress increases amount of mitochondrial RECQL4. Balance between nuclear and mitochondrial localisation regulated by interaction with p32
contribute to functions previously assigned to their mitochondrial activities [16,20]. In future, potential extramitochondrial roles will need to be considered when examining mitochondrial proteins in the context of cellular phenotypes until exclusive mitochondrial localisation has been proven. However, this can be challenging because, for some dual-targeted proteins, the amount present in one compartment is minute, making characterisation difficult [42]. The identification of new pathways of direct communication between mitochondria and nuclei expands the repertoire of mechanisms used by cells to maintain organelle homeostasis. Responses to mitochondrial activity affect all cellular functions through the regulation of energy and metabolite production; therefore, the elucidation of the underlying signalling network will have important implications for understanding metabolic disorders and ageing. [17_TD$IF]Acknowledgments We thank C. Tournier, G. Poulin, and S-H. Yang for helpful comments. The authors are funded by the Biotechnology and Biological Sciences Research Council (BB/J014834/1) and the Wellcome Trust [18_TD$IF](097820/Z/11/Z).
Trends in Biochemical Sciences, Month Year, Vol. xx, No. yy
7
TIBS 1190 No. of Pages 8
References 1. Nunnari, J. and Suomalainen, A. (2012) Mitochondria: in sickness and in health. Cell 148, 1145–1159 2. Kotiadis, V.N. et al. (2014) Mitochondrial quality control and communications with the nucleus are important in maintaining mitochondrial function and cell health. Biochim. Biophys. Acta 1840, 1254–1265 3. Chandel, N.S. (2014) Mitochondria as signaling organelles. BMC Biol. 12, 34 4. Galluzzi, L. et al. (2012) Mitochondria: master regulators of danger signalling. Nat. Rev. Mol. Cell Biol. 13, 780–788 5. Whelan, S.P. and Zuckerbraun, B.S. (2013) Mitochondrial signaling: forwards, backwards, and in between. Oxid. Med. Cell. Longev. 2013, 351613
25. McLelland, G.L. et al. (2014) Parkin and PINK1 function in a vesicular trafficking pathway regulating mitochondrial quality control. EMBO J. 33, 282–295 26. Hall, D.A. et al. (2004) Regulation of gene expression by a metabolic enzyme. Science 306, 482–484 27. Yogev, O. et al. (2010) Fumarase: a mitochondrial metabolic enzyme and a cytosolic/nuclear component of the DNA damage response. PLoS Biol. 8, e1000328 28. Yogev, O. and Pines, O. (2011) Dual targeting of mitochondrial proteins: mechanism, regulation and function. Biochim. Biophys. Acta 1808, 1012–1020 29. Yogev, O. et al. (2011) Fumarase: a paradigm of dual targeting and dual localized functions. FEBS J. 278, 4230–4242
6. Lo, S.C. and Hannink, M. (2008) PGAM5 tethers a ternary complex containing Keap1 and Nrf2 to mitochondria. Exp. Cell Res. 314, 1789–1803
30. Jiang, Y. et al. (2015) Local generation of fumarate promotes DNA repair through inhibition of histone H3 demethylation. Nat. Cell Biol. 17, 1158–1168
7. Paek, J. et al. (2012) Mitochondrial SKN-1/Nrf mediates a conserved starvation response. Cell Metab. 16, 526–537
31. Santos, J.H. et al. (2004) Mitochondrial hTERT exacerbates freeradical-mediated mtDNA damage. Aging Cell 3, 399–401
8. Itoh, K. et al. (2015) Emerging functional cross-talk between the Keap1-Nrf2 system and mitochondria. J. Clin. Biochem. Nutr. 56, 91–97
32. Chen, L-Y. et al. (2012) Mitochondrial localization of telomeric protein TIN2 links telomere regulation to metabolic control. Mol. Cell 47, 839–850
9. Nargund, A.M. et al. (2012) Mitochondrial import efficiency of ATFS-1 regulates mitochondrial UPR activation. Science 337, 587–590
33. Croteau, D.L. et al. (2012) RECQL4 localizes to mitochondria and preserves mitochondrial DNA integrity. Aging Cell 11, 456–466
10. Pellegrino, M.W. et al. (2013) Signaling the mitochondrial unfolded response. Biochim. Biophys. Acta 1833, 410–416
34. Chi, Z. et al. (2012) RecQL4 cytoplasmic localization: implications in mitochondrial DNA oxidative damage repair. Int. J. Biochem. Cell Biol. 44, 1942–1951
11. Jovaisaite, V. and Auwerx, J. (2015) The mitochondrial unfolded protein response – synchronizing genomes. Curr. Opin. Cell Biol. 33, 74–81
35. Wang, J-T. et al. (2014) Impaired p32 regulation caused by the lymphoma-prone RECQ4 mutation drives mitochondrial dysfunction. Cell Rep. 7, 848–858
12. Nargund, A.M. et al. (2015) Mitochondrial and nuclear accumulation of the transcription factor ATFS-1 Promotes OXPHOS recovery during the UPR(mt). Mol. Cell 58, 123–133
36. Ahmed, S. et al. (2008) Telomerase does not counteract telomere shortening but protects mitochondrial function under oxidative stress. J. Cell Sci. 121, 1046–1053
13. Zhao, Q. et al. (2002) A mitochondrial specific stress response in mammalian cells. EMBO J. 21, 4411–4419
37. Haendeler, J. et al. (2009) Mitochondrial telomerase reverse transcriptase binds to and protects mitochondrial DNA and function from damage. Arterioscler. Thromb. Vasc. Biol. 6, 929–935
14. Aldridge, J.E. et al. (2007) Discovery of genes activated by the mitochondrial unfolded protein response (mtUPR) and cognate promoter elements. PLoS ONE 9, e874 15. Marbois, B.N. and Clarke, C.F. (1996) The COQ7 gene encodes a protein in Saccharomyces cerevisiae necessary for ubiquinone biosynthesis. J. Biol. Chem. 271, 2995–3004 16. Monaghan, R.M. et al. (2015) A nuclear role for the respiratory enzyme CLK-1 in regulating mitochondrial stress responses and longevity. Nat. Cell Biol. 17, 782–792 17. Harbauer, A.B. et al. (2014) The protein import machinery of mitochondria - a regulatory hub in metabolism, stress and disease. Cell Metab. 19, 357–372
38. Kovalenko, O.A. et al. (2010) A mutant telomerase defective in nuclear-cytoplasmic shuttling fails to immortalize cells and is associated with mitochondrial dysfunction. Aging Cell 9, 203–219 39. Sharma, N.K. et al. (2012) Human telomerase acts as a hTRindependent reverse transcriptase in mitochondria. Nucleic Acids Res. 40, 712–725 40. Jeffery, C.J. (1999) Moonlighting proteins. Trends Biochem. Sci. 24, 8–11 41. Copley, S.D. (2012) Moonlighting is mainstream: paradigm adjustment required. Bioessays 34, 578–588
18. Lu, T.T. et al. (2013) Aging-associated enzyme human Clock-1: substrate-mediated reduction of the diiron center for 5-demethoxyubiquinone hydroxylation. Biochemistry 52, 2236–2244
42. Regev-Rudzki, N. and Pines, O. (2007) Eclipsed distribution: a phenomenon of dual targeting of protein and its significance. Bioessays 29, 772–782
19. Ploumakis, A. and Coleman, M.L. (2015) OH, the places you’ll go! Hydroxylation, gene expression, and cancer. Mol. Cell 58, 729–741
43. Youle, R.J. and Narendra, D.P. (2011) Mechanisms of mitophagy. Nat. Rev. Mol. Cell Biol. 12, 9–14
20. Sutendra, G. et al. (2014) A nuclear Pyruvate Dehydrogenase complex is important for the generation of acetyl-CoA and histone acetylation. Cell 158, 84–97
44. Wang, X. and Chen, X.J. (2015) A cytosolic network suppressing mitochondria-mediated proteostatic stress and cell death. Nature 524, 481–484
21. Chueh, F.Y. et al. (2011) Nuclear localization of pyruvate dehydrogenase complex-E2 (PDC-E2), a mitochondrial enzyme, and its role in signal transducer and activator of transcription 5 (STAT5)dependent gene transcription. Cell. Signal. 23, 1170–1178
45. Wrobel, L. et al. (2015) Mistargeted mitochondrial proteins activate a proteostatic response in the cytosol. Nature 524, 485–488
22. Patel, M.S. et al. (2014) The pyruvate dehydrogenase complexes: structure-based function and regulation. J. Biol. Chem. 289, 16615–16623 23. Cautain, B. et al. (2015) Components and regulation of nuclear transport processes. FEBS J. 282, 445–462 24. Sugiura, A. et al. (2014) A new pathway for mitochondrial quality control: mitochondrial-derived vesicles. EMBO J. 33, 2142–2156
8
Trends in Biochemical Sciences, Month Year, Vol. xx, No. yy
46. Geva, Y. and Schuldiner, M. (2014) The back and forth of cargo exit from the endoplasmic reticulum. Curr. Biol. 24, R130–R136 47. Lane, N. and Martin, W. (2010) The energetics of genome complexity. Nature 467, 929–934 48. Qattan, A.T. et al. (2012) Spatial distribution of cellular function: the partitioning of proteins between mitochondria and the nucleus in MCF7 breast cancer cells. J. Proteome Res. 11, 6080–6101
Opinion
Mitochondrial Proteins Moonlighting in the Nucleus Richard M. Monaghan1 and Alan J. Whitmarsh1,* Mitochondria function as cellular energy generators, producing the fuel required to drive biological processes. The response of cells to mitochondrial activity or dysfunction regulates their survival, growth, proliferation, and differentiation. Several proteins that contain mitochondrial-targeting sequences (MTS) also reside in the nucleus and there is increasing evidence that the nuclear translocation of mitochondrial proteins represents a novel pathway by which mitochondria signal their status to the cell. Here, we discuss the different mechanisms that control the dual mitochondrial and nuclear localisation of proteins and propose that these nuclear moonlighters represent a widespread regulatory circuit to maintain mitochondrial homeostasis. Mitochondrial Communication with the Nucleus Mitochondria host many metabolic processes, including the generation of cellular energy in the form of ATP by oxidative phosphorylation (see Glossary). Although they contain their own genome, most mitochondrial proteins are encoded in the nucleus. It is essential for nuclei to sense changes in mitochondrial metabolism and mount an appropriate response to restore homeostasis [1–3]. Disrupted communication between mitochondria and nuclei is implicated in metabolic diseases, cancer, neurodegeneration, and other ageing processes. The role of mitochondria in apoptosis is well characterised [4], but under nonapoptotic conditions, retrograde signalling pathways from mitochondria to nuclei are triggered by changes in metabolite levels or altered proteostasis [2,3,5]. Commonly, metabolites, including reactive oxygen species (ROS), act as second messengers to regulate cytoplasmic pathways [2,3,5]. However, a new paradigm is emerging whereby proteins that harbour an MTS can localise to nuclei and, in some cases, act as direct signals from mitochondria to regulate nuclear events. We propose that, by acting as signalling intermediaries between mitochondria and nuclei, these proteins promote a rapid response to changes in mitochondrial function and may directly link metabolic activity to genome integrity and gene expression.
Trends An increasing number of mitochondrial proteins have been reported to reside in the nucleus and act as mediators of direct mitochondria-to-nucleus communication. These proteins may perform similar functions in both compartments or have distinct activities. Mitochondrial protein import is responsive to mitochondrial activity and this represents a key regulatory point to determine the balance between mitochondrial and nuclear localisation. Active mitochondrial enzyme complexes may translocate from the mitochondrial matrix to nuclei. Metabolic enzymes can associate with chromatin and regulate gene expression.
Direct Mitochondria-to-Nucleus Signalling Conceptually, the translocation of mitochondrial proteins to nuclei represents the simplest and most direct means of retrograde communication between the two organelles. These proteins can be responsive to a variety of stimuli and may perform a similar function in both compartments or have distinct mitochondrial and nuclear roles. Below, we discuss some of the mechanisms involved. Nuclear Redirection of Transcription Factors in Response to Disrupted Mitochondrial Function Transcription factors can be targeted to mitochondria yet primed to be redirected to the nucleus in response to mitochondrial stress. For example, mammalian nuclear factor erythroid 2-related factor 2 (NRF2), which lacks an MTS, is sequestered in a complex with Kelch-like ECHassociated protein 1 (KEAP1) and the MTS-containing phosphatase phosphoglycerate mutase
Trends in Biochemical Sciences, Month Year, Vol. xx, No. yy
1 Faculty of Life Sciences, University of Manchester, Michael Smith Building, Oxford Road, Manchester, M13 9PT, UK
*Correspondence: [email protected] (A.J. Whitmarsh).
http://dx.doi.org/10.1016/j.tibs.2015.10.003 © 2015 Elsevier Ltd. All rights reserved.
1
TIBS 1190 No. of Pages 8
Nucleus
Glossary
Mitochondrion NRF2
PGAM5
MTS
KEAP1
Oxidave stress
ATFS-1 Disrupted proteostasis
MTS
ATFS-1
MTS
NRF2 Anoxidant response
ATFS-1
UPRmt
Histone acetylaon
CLK-1
MTS
ROS barometer
mtDNA
MTS
CLK-1
OXPHOS
CLK-1
Acetyl-CoA
Acetyl-CoA Growth signals PDC
ETS inhibion PDC proteins
MTS
PDC
Figure 1. Mitochondrial Proteins Directly Signal to the Nucleus to Regulate Gene Expression. In mammalian cells, the transcription factor NRF2 associates with the outer mitochondrial membrane as part of a complex with KEAP1 and PGAM5, but dissociates from the complex upon oxidative stress and translocates to the nucleus, where it targets the promoters of genes that contribute to antioxidant defences [6–8]. The Caenorhabditis elegans transcription factor ATFS-1 is imported into the mitochondrial matrix and degraded by proteolysis [9], while CLK-1 and components of the PDC are imported into mitochondria and their N-terminal MTSs cleaved [16,20]. CLK-1 is required for the synthesis of ubiquinone and PDC generates acetyl-CoA, both of which contribute to oxidative phosphorylation (OXPHOS) [15,22]. ATFS-1 and CLK-1 are redirected from mitochondria to nuclei in response to disrupted proteostasis and [7_TD$IF]physiological ROS [8_TD$IF]production, respectively [9,16]. ATFS-1 mediates the UPRmt and the expression of mitochondrial-encoded genes in C. elegans [9,12], while CLK-1 acts to limit stress responses in both C. elegans and human cells [16]. PDC is proposed to translocate from mitochondria to nuclei as an intact complex in human cells in response to growth signals or impaired oxidative phosphorylation. Similar to its mitochondrial activity, nuclear PDC generates acetyl-CoA and this is used as a co-factor for histone acetylation [20]. Abbreviations: ATFS-1, activating transcription factor associated with stress 1; CLK-1, CLOCK-1; CoA, coenzyme A; ETS, electron transport system; KEAP1, Kelch-like ECH-associated protein 1; MTS, mitochondrial-targeting sequence; NRF2, nuclear factor erythroid 2-related factor 2; OXPHOS, oxidative phosphorylation; PDC, pyruvate dehydrogenase complex; PGAM5, phosphoglycerate mutase 5; ROS, reactive oxygen species.
5 (PGAM5) at the outer mitochondrial membrane [6,7]. Upon mitochondrial stress, NRF2 dissociates from the complex and translocates to the nucleus to activate its target genes [6] (Figure 1). This close proximity with the primary site of cellular ROS production allows NRF2 to rapidly mobilise antioxidant defences when mitochondrial oxidative stress occurs [8]. In contrast to NRF2, the nuclear localisation of the Caenorhabditis elegans transcription factor Activating Transcription Factor associated with Stress 1 (ATFS-1) is regulated by the efficiency of its mitochondrial import [9]. ATFS-1 is a component of the mitochondrial unfolded protein response (UPRmt[14_TD$IF]), which is triggered by disrupted proteostasis [10,11]. Normally, ATFS-1 is imported into mitochondria, where it undergoes proteolytic degradation; however, upon UPRmt induction, it is stabilised within the mitochondria and interacts with the mitochondrial genome to limit the accumulation of mitochondrially encoded mRNAs [9,12]. Concurrently, mitochondrial import of ATFS-1 is impaired and a pool is redirected to the nucleus, where it regulates genes encoding proteins required for maintaining proteostasis and oxidative phosphorylation [9,12] (Figure 1). ATFS-1 is able to localise to both organelles because it contains an MTS and a nuclear localisation signal; it is the coordinated mitochondrial stabilisation and nuclear localisation of
2
Trends in Biochemical Sciences, Month Year, Vol. xx, No. yy
Echoforms: identical forms of a protein at different subcellular locations. Eclipsed distribution: the uneven distribution of a protein between two cellular compartments. Endosymbiosis: the theory that organelles, such as mitochondria, originated as a symbiosis between two distinct prokaryotic cells. Homeostasis: the maintenance of a constant internal environment in cells or organisms to maintain functioning by compensating for changing conditions. Mitochondrial targeting sequence (MTS): a short amino acid sequence usually found near the N terminus of nuclear-encoded mitochondrial proteins that can interact with the import machinery located within the inner and outer mitochondrial membranes. Mitochondrial unfolded protein response (UPRmt): a mitochondriato-nucleus signalling pathway that responds to mitochondrial stress, particularly the accumulation of misfolded or unfolded proteins in mitochondria or an imbalance between mitochondrial and nuclear encoded proteins. Mitophagy: the selective degradation of damaged mitochondria by autophagy. Moonlighting protein: a protein with more than one function. Oxidative phosphorylation (OXPHOS): the process by which electrons are transferred to oxygen by the electron transport chain located at the mitochondrial inner membrane to produce energy in the form of ATP. Proteostasis: the coordinated regulation of proteins including their translation, folding, movement, and degradation. Cells try to maintain proteostasis, particularly in response to stress. Reactive oxygen species (ROS): chemically reactive molecules that contain oxygen. Mitochondria are the major site of ROS generation in cells because ROS are a by-product of oxidative phosphorylation. Retrograde signalling: in the context of this article, this refers to signalling from mitochondria to the nucleus. Signalling from the nucleus to other organelles is termed anterograde signalling.
TIBS 1190 No. of Pages 8
ATFS-1 that allows it to act as a sensor of mitochondrial stress and promote the recovery of oxidative phosphorylation [9,12]. The UPRmt is a conserved retrograde pathway in eukaryotes [10,11], but is relatively poorly characterised in mammals. The transcription factors C/EBP homologous protein (CHOP) and CCAAT-enhancer binding protein b (C/EBPb) have been proposed to coordinate mammalian UPRmt target gene expression, but they are not direct signalling intermediaries between mitochondria and nuclei [13,14]. Therefore, it will be interesting to determine whether functional homologues of ATFS-1 exist in mammalian cells. Maintaining Mitochondrial Homeostasis It is important that the stress-response programmes, such as those mediated by NRF2 and ATFS-1, are carefully monitored. The mitochondrial enzyme CLOCK-1 (CLK-1; also called COQ7) may have a key role in this. CLK-1 catalyses a step in the biosynthesis of ubiquinone, a component of the electron transport system [15], and was recently shown to reside in nuclei and regulate gene expression in both C. elegans and human cells [16]. CLK-1 is present in the nucleus in the absence of mitochondrial stress, but its localisation is dependent on physiological ROS production [16]. Importantly, nuclear CLK-1 suppresses gene expression mediated by ROS-induced stress pathways and the UPRmt, and so it may function to limit the activation of a major stress response until a particular threshold of mitochondrial dysfunction is reached [16]. In this way, the nuclear translocation of CLK-1 acts as a ROS barometer to maintain mitochondrial homeostasis (Figure 1).
Retrotranslocation: in the context of this article, the movement of a protein out of mitochondria contrary to its normal route of import and processing. Second messengers: intracellular signalling molecules released within cells to initiate signal transduction cascades. Tricarboxylic acid (TCA) cycle: a metabolic pathway found in mitochondria that releases electrons to the electron transport chain for oxidative phosphorylation. It also provides the building blocks for the biosynthesis of amino acids, fatty acids, and other biological molecules.
The exact mechanism by which CLK-1 and ATFS-1 are redirected to the nucleus remains unclear. The nuclear forms retain their MTS, indicating that they have not been imported into the mitochondrial matrix (where their MTS would be cleaved by proteases [17]). However, this would not exclude them being initially targeted to the outer mitochondrial membrane or translocated into the intermembrane space before their nuclear translocation. The mechanism by which nuclear CLK-1 regulates gene expression is also not known. CLK-1 is an iron-binding monooxygenase that has hydroxylase activity [18], and it will be important to ascertain whether this activity is required for its nuclear role. If so, given that CLK-1 associates with chromatin [16], it is possible that it could directly modify DNA or histones. While this would be a novel role for a monooxygenase, it is noteworthy that iron-binding dioxygenases have been shown to regulate gene expression through the demethylation of cytosine bases and histones [19]. Double Duty for a Mitochondrial Matrix Complex In contrast to ATFS-1 and CLK-1, the human pyruvate dehydrogenase complex (PDC) appears to translocate directly from the mitochondrial matrix to the nucleus [20]. This is supported by the observation that nuclear PDC components lack their MTS, implying prior processing in the mitochondrial matrix [20,21]. PDC links cytoplasmic glycolytic metabolism to mitochondrial oxidative phosphorylation by converting pyruvate to the tricarboxylic acid (TCA) cycle substrate acetyl co-enzyme A (-CoA) [22]. It performs the same enzymatic function in the nucleus, providing a pool of acetyl-CoA that is used as a cofactor for histone acetylation [20]. Nuclear PDC levels are cell cycle dependent and increase in response to growth signals or the inhibition of oxidative phosphorylation [20]. Therefore, PDC coordinates mitochondrial metabolism with nuclear gene expression to regulate cell growth (Figure 1). The proposal that PDC translocates from the mitochondrial matrix to the nucleus as an intact complex is intriguing. To do this, PDC must traverse both mitochondrial membranes before moving to the nucleus. The nuclear import of large complexes is well characterised [23], but their mitochondrial export is not. Mitochondrial vesicles have recently been identified as a novel mechanism to selectively degrade discrete regions of mitochondrial membranes and associated proteins [24]. PDC components have been detected in these [25], so it is inviting to speculate that a nondegradatory vesicular pathway exists in which proteins destined for locations
Trends in Biochemical Sciences, Month Year, Vol. xx, No. yy
3
TIBS 1190 No. of Pages 8
secondary to the mitochondria, for example the nucleus, are redirected. Another possibility is that PDC components retrotranslocate back out of the mitochondria following cleavage of their N-terminal MTS and form a complex in the cytosol that translocates to the nucleus. Alternatively, leakage or breakdown of the mitochondrial membrane could occur, even under nonapoptotic conditions, allowing proteins to exit from the mitochondrial matrix.
Dual Targeting to Mitochondria and Nuclei The proteins discussed so far directly link mitochondrial activity to nuclear events. However, for other MTS-containing proteins that are targeted to both compartments, it is less clear whether the two protein pools communicate with each other or act separately. For example, the yeast enzyme Arg5,6 is required for arginine biosynthesis in mitochondria and regulates nuclear gene expression, but whether it integrates these two processes is not known [26]. In some cases, these proteins may constitute an anterograde pathway from the nucleus to mitochondria. Alternatively, regulating the relative amounts of the protein in each compartment may indirectly affect the function of the other organelle. Dual Targeting of Fumarase One of the best-characterised dual-targeted proteins is the enzyme fumarase, which participates in the TCA cycle in mitochondria and regulates the response to DNA damage in the nucleus [27] (Figure 2). The two pools of fumarase have been coined [3_TD$IF]echoforms[4_TD$IF], a term applicable to identical forms of a protein that are found at different cellular locations [28]. The respective rates of protein translation and mitochondrial import control the mitochondrial
Nucleus
Mitochondrion
Telomere maintenance
TIN2
MTS
MTS
TPP1 TIN2
TIN2
TERT
MTS
MTS
TERT
MTS
ROS TERT
mtDNA
RECQL4
RECQL4
p32
Fumarase
Fumarase
TCA cycle
MTS
Fumarase
Fumarase
MTS
RECQL4
MTS
DNA damage response
MTS
ROS
Figure 2. Dual-Targeted Proteins Maintain Nuclear and Mitochondrial Genome Integrity in Response to Stress. Several regulators of nuclear genome stability contain an MTS and are targeted to mitochondria. In mammalian cells, mitochondrial TERT and RECQL4 levels are enhanced by increased ROS, while the balance of nuclear and mitochondrial localisation of both TIN2 and RECQL4 can be regulated by their binding partners, TPP1 and p32, respectively [32–36]. In mitochondria, TERT and RECQL4 help maintain mitochondrial genome integrity and copy number, and protect from ROS stress. TIN2 regulates mitochondrial morphology and promotes ROS production [32]. The TCA cycle enzyme fumarase participates in the nuclear DNA damage response and its localisation in yeast is, in part, controlled by the comparative rates of its mitochondrial import and protein translation [27,29]. Abbreviations: mtDNA, mitochondrial DNA; MTS, mitochondrial-targeting sequence; RECQL4, RecQ helicase-like 4; ROS, reactive oxygen species; TCA, tricarboxylic acid; TERT, telomerase reverse transcriptase; TIN2, TERF1-interacting protein 2; TPP1, [9_TD$IF]TIN2 [10_TD$IF]interacting protein 1.
4
Trends in Biochemical Sciences, Month Year, Vol. xx, No. yy
TIBS 1190 No. of Pages 8
localisation of fumarase in yeast [29]. Fumarase is imported co-translationally; if import slows, it starts to fold in the cytoplasm, thus blocking its further import and resulting in its retrotranslocation back into the cytoplasm [29]. In contrast to ATFS-1 and CLK-1, the N-terminal MTS of fumarase reaches the mitochondrial matrix and is cleaved before retrotranslocation, so both cytosolic and mitochondrial fumarase are the processed forms of the enzyme [29]. In response to DNA double-strand breaks, fumarase is recruited from the cytosol to the nucleus, where it produces fumarate, which contributes to regulating the DNA damage response in both yeast and human cells [27]. Recent evidence suggests that nuclear fumarate promotes DNA repair by inhibiting histone demethylation [30]. Interestingly, the mammalian homologue of fumarase appears to be regulated differently; distinct translation products that are produced from a single gene are differentially localised [29]. Regulating Nuclear and Mitochondrial Genome Stability There are other proteins that are required for maintaining mammalian genome integrity, contain an N-terminal MTS, and display dual mitochondrial and nuclear localisation. These include the telomerase component telomerase reverse transcriptase (TERT), which prevents telomere shortening in the nucleus; the telomeric protein TERF1-interacting protein 2 (TIN2); and the DNA helicase RecQ helicase-like 4 (RECQL4) [31–35] (Figure 2). In mitochondria, TERT protects the mitochondrial genome from oxidative stress [36–39], TIN2 is proposed to regulate oxidative phosphorylation and mitochondrial morphology [32], and RECQL4 controls mitochondrial DNA copy number and integrity [33–35]. The N terminus of TIN2 is processed in the mitochondria, but it is unclear whether this is the case for TERT or RECQL4. The nuclear and mitochondrial pools of these proteins appear to act separately, but the balance between them can be regulated. For example, TERT is predominantly nuclear, but oxidative stress can shift it to be predominantly mitochondrial, indicative of anterograde signalling from nuclei to mitochondria [36]. This suggests that protecting the mitochondrial genome is prioritised over the nuclear genome under these conditions. It is likely that redirecting resources to protect the mitochondrial genome is a survival strategy, because mitochondrial dysfunction can lead to increased local levels of ROS that could damage the nearby mitochondrial DNA.
Advantages of Dual Targeting and Direct Mitochondrial to Nuclear Signalling The targeting of the same protein to different cellular locations appears to be a widespread phenomenon that operates alongside other mechanisms, including alternative splicing or alternative translation, to limit the requirement for gene duplication. Combined with the increasing number of proteins that have been identified as having more than one function [28,40,41], this suggests that there is evolutionary pressure for proteins to acquire additional cellular roles. An advantage of this would be the efficient management of resources. It is evident that many dual-targeted proteins have an eclipsed distribution, meaning that they predominantly localise and function in one compartment, with only a small pool required in the second compartment [42]. Depending on need, the distribution of the protein between compartments may be rapidly modulated and, in some cases, the translocation of the protein between compartments acts as a signal that allows functional integration. For those proteins that translocate from mitochondria to the nucleus, what are the advantages of this direct rerouting compared with signalling through cytosolic pathways? Perhaps by associating with the outer membrane or being integral mitochondrial proteins, they can respond to changes in metabolic activity that secondary messenger levels in the cytoplasm are unable to communicate. Indeed, some responses may only be possible from within mitochondria; for example, [15_TD$IF]in reaction to changes at the inner membrane where oxidative phosphorylation occurs. Of note, the inhibition of mitochondrial protein import may be a common mechanism by which mitochondrial activity is monitored by cells. For example, the impaired mitochondrial import of ATFS-1 and CLK-1 is sufficient to redirect them to the nucleus [9,16]. Also, the selective
Trends in Biochemical Sciences, Month Year, Vol. xx, No. yy
5
TIBS 1190 No. of Pages 8
degradation of mitochondria (mitophagy) and a newly characterised stress pathway in yeast, activated by cytosolic accumulation of toxic mitochondrial precursor proteins, are both mediated by changes to mitochondrial protein import [43–45]. Therefore, it is plausible that a range of mitochondrial states can be communicated by the ability of different proteins to be imported. The import of mitochondrial proteins assists in their folding and integration into complexes [17], and so mitochondrial import may be required as a preceding step to form active proteins before their translocation to the nucleus. This could be the case for PDC, which is proposed to translocate as an active complex to nuclei from the mitochondrial matrix [20]. This would be somewhat analogous to the well-characterised endoplasmic reticulum pathway of protein folding and maturation, and the subsequent transport of proteins to distinct cellular localisations [46]. Bearing in mind that mitochondria originated as a result of endosymbiosis [47], it is conceivable that appropriate conditions for particular proteins to fold and form complexes may uniquely exist within mitochondria and that no alternate cellular environment evolved to fulfil a similar role. Thus, if an extramitochondrial function arose later in evolution, this could only be achieved by translocation from mitochondria following import and correct folding. One consequence of direct signalling from individual mitochondrial sites is that it permits a localised stress to elicit a retrograde response. In such circumstances, a global change in nuclear gene expression would not necessarily be warranted. Therefore, controlling the balance of the activities of different mitochondria-to-nuclear translocation pathways is important. If most mitochondria are functioning normally, this would maintain a pool of nuclear CLK-1 that could dampen retrograde stress responses mediated by NRF2 or ATFS-1 and triggered by a few dysfunctional mitochondria. These mitochondria could be pushed towards mitophagy and degradation [43], rather than mounting a protective programme of gene expression. In this way, the integration of antagonistic pathways that are sensitive to various levels of mitochondrial stress would dictate the response mounted to maintain mitochondrial and cellular homeostasis. A further consequence of[5_TD$IF] translocation[6_TD$IF] away from mitochondria to the nucleus would be to decrease the mitochondrial pool[16_TD$IF] of proteins. Relatively small reductions in the levels of the mitochondrial proteins may not appear relevant for abundant metabolic proteins, such as CLK-1 and PDC, but at the level of an individual mitochondrion they could be. Indeed, autoregulatory loops may operate, whereby redirection to the nucleus decreases the mitochondrial protein pool and directly alters aspects of mitochondrial function. This could lead to inhibition of nuclear localisation and/or promote mitochondrial import of the protein, thereby re-establishing the mitochondrial function. Such a mechanism could operate in tandem with the global response mounted in the nucleus that may reverse the original signal. The integration of multiple regulatory strands would act as a sensitive rheostat of mitochondrial fitness.
Concluding Remarks Considering the importance of maintaining mitochondrial integrity and optimal metabolism for cellular fitness, it is unsurprising that many signalling routes between mitochondria and the nucleus have evolved. The recent studies uncovering direct signalling intermediaries that functionally link these compartments has shifted perceptions of how mitochondria signal (Table 1). Clearly, further investigation into the distinct mechanisms involved is warranted. Importantly, several hundred proteins have been reported to display both mitochondrial and nuclear localisation [48]. A number of these harbour an MTS, but their nuclear roles remain largely uncharacterised (see Outstanding Questions). However, it points to the nuclear moonlighting of mitochondrial proteins being part of an extensive signalling network within cells. This has implications for interpreting studies of proteins previously assumed to be exclusively mitochondrial, because their nuclear roles may contribute to reported functions and associated phenotypes. This is true for PDC and CLK-1, where their nuclear roles have now been shown to
6
Trends in Biochemical Sciences, Month Year, Vol. xx, No. yy
Outstanding Questions What is the full extent of mitochondrial proteins moonlighting in the nucleus? Many proteins have been reported to localise to both compartments and several contain an MTS. It will be important to characterise their nuclear functions. Can the functions and associated phenotypes of mitochondrial proteins, previously presumed to reflect their mitochondrial role, be explained by their newly discovered nuclear roles? How are MTS-containing mitochondrial proteins, such as CLK-1 and ATFS-1, redirected to nuclei? The regulation of mitochondrial import efficiency is clearly important, but the molecular mechanisms involved need to be resolved. How do mitochondrial enzyme complexes, such as PDC, translocate to the nucleus? There is currently no mechanism to explain how a large active enzyme complex can move from the mitochondrial matrix to the nucleus. How do metabolic enzymes regulate gene transcription? CLK-1 and Arg5,6 do not have obvious DNA binding or transcription activation and/or repression domains, so how they alter transcription is unclear. Do they associate with specific transcription factors or modify chromatin? Is their enzymatic activity required?
TIBS 1190 No. of Pages 8
Table 1. MTS-Containing Proteins with Dual Mitochondrial and Nuclear Functions Protein
Species
Mitochondrial Function
Nuclear Function
Interaction between Two Organelles
ATFS-1
Caenorhabditis elegans
Limits mitochondrial mRNA production
Mediates UPRmt[1_TD$IF] by direct regulation of gene expression
Disrupted mitochondrial proteostasis stabilises mitochondrial ATFS-1 and directs pool of protein to nucleus
CLK-1
C. elegans and human
Ubiquinone biosynthesis
Modulates mitochondrial stress responses through regulating gene expression
Localises predominantly to mitochondria with a small amount redirected to nucleus in response to physiological ROS production
PDC
Human
Acetyl-CoA production for the TCA cycle
Acetyl-CoA production for histone acetylation
Translocates from mitochondria to nuclei in response to growth signals or impaired oxidative phosphorylation
Fumarase
Yeast
Fumarate production in TCA cycle
Fumarate production for DNA damage response
Cleaved during mitochondrial import with the nuclear form retrotranslocating from mitochondria
TERT
Human
Protects from telomere shortening
Protects mtDNA from oxidative damage
Oxidative stress increases amount of mitochondrial TERT
TIN2
Human
Regulation of telomeres
Regulates oxidative phosphorylation
Balance between nuclear and mitochondrial localisation regulated by interaction with [9_TD$IF]TIN2 [1_TD$IF]interacting protein 1 [12_TD$IF](TPP1[13_TD$IF])
RECQL4
Human
Maintaining genome stability
Controls mtDNA copy number and integrity
Oxidative stress increases amount of mitochondrial RECQL4. Balance between nuclear and mitochondrial localisation regulated by interaction with p32
contribute to functions previously assigned to their mitochondrial activities [16,20]. In future, potential extramitochondrial roles will need to be considered when examining mitochondrial proteins in the context of cellular phenotypes until exclusive mitochondrial localisation has been proven. However, this can be challenging because, for some dual-targeted proteins, the amount present in one compartment is minute, making characterisation difficult [42]. The identification of new pathways of direct communication between mitochondria and nuclei expands the repertoire of mechanisms used by cells to maintain organelle homeostasis. Responses to mitochondrial activity affect all cellular functions through the regulation of energy and metabolite production; therefore, the elucidation of the underlying signalling network will have important implications for understanding metabolic disorders and ageing. [17_TD$IF]Acknowledgments We thank C. Tournier, G. Poulin, and S-H. Yang for helpful comments. The authors are funded by the Biotechnology and Biological Sciences Research Council (BB/J014834/1) and the Wellcome Trust [18_TD$IF](097820/Z/11/Z).
Trends in Biochemical Sciences, Month Year, Vol. xx, No. yy
7
TIBS 1190 No. of Pages 8
References 1. Nunnari, J. and Suomalainen, A. (2012) Mitochondria: in sickness and in health. Cell 148, 1145–1159 2. Kotiadis, V.N. et al. (2014) Mitochondrial quality control and communications with the nucleus are important in maintaining mitochondrial function and cell health. Biochim. Biophys. Acta 1840, 1254–1265 3. Chandel, N.S. (2014) Mitochondria as signaling organelles. BMC Biol. 12, 34 4. Galluzzi, L. et al. (2012) Mitochondria: master regulators of danger signalling. Nat. Rev. Mol. Cell Biol. 13, 780–788 5. Whelan, S.P. and Zuckerbraun, B.S. (2013) Mitochondrial signaling: forwards, backwards, and in between. Oxid. Med. Cell. Longev. 2013, 351613
25. McLelland, G.L. et al. (2014) Parkin and PINK1 function in a vesicular trafficking pathway regulating mitochondrial quality control. EMBO J. 33, 282–295 26. Hall, D.A. et al. (2004) Regulation of gene expression by a metabolic enzyme. Science 306, 482–484 27. Yogev, O. et al. (2010) Fumarase: a mitochondrial metabolic enzyme and a cytosolic/nuclear component of the DNA damage response. PLoS Biol. 8, e1000328 28. Yogev, O. and Pines, O. (2011) Dual targeting of mitochondrial proteins: mechanism, regulation and function. Biochim. Biophys. Acta 1808, 1012–1020 29. Yogev, O. et al. (2011) Fumarase: a paradigm of dual targeting and dual localized functions. FEBS J. 278, 4230–4242
6. Lo, S.C. and Hannink, M. (2008) PGAM5 tethers a ternary complex containing Keap1 and Nrf2 to mitochondria. Exp. Cell Res. 314, 1789–1803
30. Jiang, Y. et al. (2015) Local generation of fumarate promotes DNA repair through inhibition of histone H3 demethylation. Nat. Cell Biol. 17, 1158–1168
7. Paek, J. et al. (2012) Mitochondrial SKN-1/Nrf mediates a conserved starvation response. Cell Metab. 16, 526–537
31. Santos, J.H. et al. (2004) Mitochondrial hTERT exacerbates freeradical-mediated mtDNA damage. Aging Cell 3, 399–401
8. Itoh, K. et al. (2015) Emerging functional cross-talk between the Keap1-Nrf2 system and mitochondria. J. Clin. Biochem. Nutr. 56, 91–97
32. Chen, L-Y. et al. (2012) Mitochondrial localization of telomeric protein TIN2 links telomere regulation to metabolic control. Mol. Cell 47, 839–850
9. Nargund, A.M. et al. (2012) Mitochondrial import efficiency of ATFS-1 regulates mitochondrial UPR activation. Science 337, 587–590
33. Croteau, D.L. et al. (2012) RECQL4 localizes to mitochondria and preserves mitochondrial DNA integrity. Aging Cell 11, 456–466
10. Pellegrino, M.W. et al. (2013) Signaling the mitochondrial unfolded response. Biochim. Biophys. Acta 1833, 410–416
34. Chi, Z. et al. (2012) RecQL4 cytoplasmic localization: implications in mitochondrial DNA oxidative damage repair. Int. J. Biochem. Cell Biol. 44, 1942–1951
11. Jovaisaite, V. and Auwerx, J. (2015) The mitochondrial unfolded protein response – synchronizing genomes. Curr. Opin. Cell Biol. 33, 74–81
35. Wang, J-T. et al. (2014) Impaired p32 regulation caused by the lymphoma-prone RECQ4 mutation drives mitochondrial dysfunction. Cell Rep. 7, 848–858
12. Nargund, A.M. et al. (2015) Mitochondrial and nuclear accumulation of the transcription factor ATFS-1 Promotes OXPHOS recovery during the UPR(mt). Mol. Cell 58, 123–133
36. Ahmed, S. et al. (2008) Telomerase does not counteract telomere shortening but protects mitochondrial function under oxidative stress. J. Cell Sci. 121, 1046–1053
13. Zhao, Q. et al. (2002) A mitochondrial specific stress response in mammalian cells. EMBO J. 21, 4411–4419
37. Haendeler, J. et al. (2009) Mitochondrial telomerase reverse transcriptase binds to and protects mitochondrial DNA and function from damage. Arterioscler. Thromb. Vasc. Biol. 6, 929–935
14. Aldridge, J.E. et al. (2007) Discovery of genes activated by the mitochondrial unfolded protein response (mtUPR) and cognate promoter elements. PLoS ONE 9, e874 15. Marbois, B.N. and Clarke, C.F. (1996) The COQ7 gene encodes a protein in Saccharomyces cerevisiae necessary for ubiquinone biosynthesis. J. Biol. Chem. 271, 2995–3004 16. Monaghan, R.M. et al. (2015) A nuclear role for the respiratory enzyme CLK-1 in regulating mitochondrial stress responses and longevity. Nat. Cell Biol. 17, 782–792 17. Harbauer, A.B. et al. (2014) The protein import machinery of mitochondria - a regulatory hub in metabolism, stress and disease. Cell Metab. 19, 357–372
38. Kovalenko, O.A. et al. (2010) A mutant telomerase defective in nuclear-cytoplasmic shuttling fails to immortalize cells and is associated with mitochondrial dysfunction. Aging Cell 9, 203–219 39. Sharma, N.K. et al. (2012) Human telomerase acts as a hTRindependent reverse transcriptase in mitochondria. Nucleic Acids Res. 40, 712–725 40. Jeffery, C.J. (1999) Moonlighting proteins. Trends Biochem. Sci. 24, 8–11 41. Copley, S.D. (2012) Moonlighting is mainstream: paradigm adjustment required. Bioessays 34, 578–588
18. Lu, T.T. et al. (2013) Aging-associated enzyme human Clock-1: substrate-mediated reduction of the diiron center for 5-demethoxyubiquinone hydroxylation. Biochemistry 52, 2236–2244
42. Regev-Rudzki, N. and Pines, O. (2007) Eclipsed distribution: a phenomenon of dual targeting of protein and its significance. Bioessays 29, 772–782
19. Ploumakis, A. and Coleman, M.L. (2015) OH, the places you’ll go! Hydroxylation, gene expression, and cancer. Mol. Cell 58, 729–741
43. Youle, R.J. and Narendra, D.P. (2011) Mechanisms of mitophagy. Nat. Rev. Mol. Cell Biol. 12, 9–14
20. Sutendra, G. et al. (2014) A nuclear Pyruvate Dehydrogenase complex is important for the generation of acetyl-CoA and histone acetylation. Cell 158, 84–97
44. Wang, X. and Chen, X.J. (2015) A cytosolic network suppressing mitochondria-mediated proteostatic stress and cell death. Nature 524, 481–484
21. Chueh, F.Y. et al. (2011) Nuclear localization of pyruvate dehydrogenase complex-E2 (PDC-E2), a mitochondrial enzyme, and its role in signal transducer and activator of transcription 5 (STAT5)dependent gene transcription. Cell. Signal. 23, 1170–1178
45. Wrobel, L. et al. (2015) Mistargeted mitochondrial proteins activate a proteostatic response in the cytosol. Nature 524, 485–488
22. Patel, M.S. et al. (2014) The pyruvate dehydrogenase complexes: structure-based function and regulation. J. Biol. Chem. 289, 16615–16623 23. Cautain, B. et al. (2015) Components and regulation of nuclear transport processes. FEBS J. 282, 445–462 24. Sugiura, A. et al. (2014) A new pathway for mitochondrial quality control: mitochondrial-derived vesicles. EMBO J. 33, 2142–2156
8
Trends in Biochemical Sciences, Month Year, Vol. xx, No. yy
46. Geva, Y. and Schuldiner, M. (2014) The back and forth of cargo exit from the endoplasmic reticulum. Curr. Biol. 24, R130–R136 47. Lane, N. and Martin, W. (2010) The energetics of genome complexity. Nature 467, 929–934 48. Qattan, A.T. et al. (2012) Spatial distribution of cellular function: the partitioning of proteins between mitochondria and the nucleus in MCF7 breast cancer cells. J. Proteome Res. 11, 6080–6101