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ScienceDirect Metabolic control via the mitochondrial protein import machinery Magdalena Opalin´ska1 and Chris Meisinger1,2 Mitochondria have to import most of their proteins in order to fulfill a multitude of metabolic functions. Sophisticated import machineries mediate targeting and translocation of preproteins from the cytosol and subsequent sorting into their suborganellar destination. The mode of action of these machineries has been considered for long time as a static and constitutively active process. However, recent studies revealed that the mitochondrial protein import machinery is subject to intense regulatory mechanisms that include direct control of protein flux by metabolites and metabolic signalling cascades. Addresses 1 Institut fu¨r Biochemie und Molekularbiologie, ZBMZ, Germany 2 BIOSS Centre for Biological Signalling Studies, Germany Corresponding author: Meisinger, Chris ([email protected]
Current Opinion in Cell Biology 2015, 33:42–48 This review comes from a themed issue on Cell regulation Edited by Johan Auwerx and Jodi Nunnari
are central integrators of cellular metabolism by supplying energy and essential building blocks necessary for lifesustaining biochemical reactions. The complexity and indispensability of these functions have positioned mitochondria on the crossroad of various signalling cascades and made them key mediators of cell death [2,9–12]. Consequently, to maintain their integrity, cells have to adjust mitochondrial activity in response to changes in energetic demands and supply. The adaptation of capacity and efficiency of ATP production by mitochondria is a very complex process, which is achieved through the interplay of diverse mechanisms [13–15,16,17,18–21]. Numerous signalling circuits are engaged in sensing metabolic changes and in modulation of mitochondrial biogenesis and function, for example, by altering expression and translation of mitochondrial-related genes, by influencing mitochondrial dynamics or by adjusting the activity of mitochondrial enzymes [13–15,16,17,18–21]. This minireview addresses recent findings that reveal dynamic tuning of the mitochondrial protein import machinery in order to integrate energy consumption requirements with the availability of metabolic supplies.
http://dx.doi.org/10.1016/j.ceb.2014.11.001 0955-0674/# 2014 Elsevier Ltd. All right reserved.
Introduction The continuous supply of energy in form of ATP is indispensable to sustain cellular function and viability in all living organisms. Almost all eukaryotic cells possess unique organelles, mitochondria, which have a central role in energy metabolism. These organelles originated approximately 1.5–2 billion years ago, when mitochondrial ancestors, a-proteobacteria, started an exceptional symbiotic relationship with primordial eukaryotic cells. Since then, eukaryotic cells have acquired the ability to survive under aerobic conditions via a metabolic process called oxidative phosphorylation (OXPHOS) by which the bulk of cellular ATP is produced [1,2]. Noteworthy, mitochondria are not just the mere ‘powerhouses’ of the cell. The mitochondrial proteome consists of up to 1000 (yeast) to 1500 (human) proteins, of which a variety are engaged in many metabolic pathways [3–6]. For instance, mitochondria have essential roles in the metabolism of sugars, amino acids and lipids or in the biosynthesis of cofactors like iron-sulphur clusters and heme [6–8]. Thus, mitochondria Current Opinion in Cell Biology 2015, 33:42–48
Molecular mechanisms of mitochondrial protein trafficking Though mitochondria possess their own genome and translation machinery, but only a small number of mitochondrial proteins, including a few core constituents of the respiratory chain complexes, are encoded by mtDNA and synthetized within the organelle. Thus, the vast majority of mitochondrial proteins are nuclear-encoded and have to be imported into the organelle. Upon synthesis on free ribosomes, mitochondrial precursor proteins reach the surface of the organelle in a process that is guided by cytosolic chaperones. Subsequently, they are imported by specialized protein import machineries and sorted to the designated submitochondrial destination in the outer membrane, inner membrane, intermembrane space or matrix [6,22,23]. Import and sorting of mitochondrial precursors is facilitated by cleavable N-terminal extensions (presequences) or by internal targeting sequences. These signals are recognized by specialized import receptors at the mitochondria. The translocase of the outer mitochondrial membrane (TOM complex) provides the central protein entry gate for virtually all precursor proteins. Its central component is the b-barrel protein Tom40 that serves as translocation channel for precursors across the outer membrane. Targeting signals of most incoming precursors are initially recognized at the www.sciencedirect.com
Regulation of mitochondrial protein import machinery Opalin´ska and Meisinger 43
TOM complex by the import receptors Tom70 and Tom20, membrane-anchored proteins with cytosolexposed domains. Tom20 preferentially binds precursors with a cleavable N-terminal presequence while Tom70 recognizes hydrophobic preproteins such as the metabolite carriers of the inner membrane. Precursors are then delivered to the central import receptor Tom22 and subsequently passed through the Tom40 channel [24–27]. The three small subunits Tom5, Tom6 and Tom7 are also core components of the TOM complex and function in the maintenance of its architecture. After passage through the TOM complex, preproteins are delivered to the destined submitochondrial location by specialized sorting pathways (Figure 1; see Refs. [22–27] for detailed reviews on mitochondrial protein sorting).
Mitochondrial protein import is tightly tuned to meet metabolic demands: lessons from yeast A gradual stream of reports has revealed that trafficking of nuclear-encoded mitochondrial precursors is controlled by metabolic requirements. This is illustrated for example, by the alternating subcellular distribution of fumarase in response to metabolic changes (Figure 2a). In yeast, fumarase is dually localized. It is a key enzyme of the TCA cycle in mitochondria and the glyoxylate shunt
in the cytosol. Both, the mitochondrial and the cytosolic forms of fumarase are derived from the same precursor and both are processed by the mitochondrial processing peptidase in the matrix, that removes the presequence of the enzyme. The cytosolic form is then released by retrograde translocation. The competition between fumarase folding in the cytosol and the rate of its import into the organelle seems to be a central sorting factor . Inhibition of the glyoxylate shunt, for example, by the addition of the end product of the pathway, seems to favor its mitochondrial localization. When both glyoxylate shunt and TCA cycle are active, fumarase is redistributed between cytosol and mitochondria . Although the molecular mechanism behind this phenomenon remains to be elucidated, it appears that the capacity of the mitochondrial protein import machinery to translocate fumarase precursors is adjusted to the metabolic situation in the cell. Similarly, the import of 5-aminolevulinate synthase, an enzyme that catalyzes the first and ratelimiting step of heme biosynthesis in the mitochondrial matrix, is coupled to metabolic requirements (Figure 2b). Binding of the end product heme to the 5-aminolevulinate synthase precursor in the cytosol seems to impair interaction with the import receptors and therefore inhibits its import into mitochondria [30,31,32]. Therefore, the mitochondrial protein import machinery might play
cysteine-rich IMS protein precursor
cleavable precursor of matrix or IM proteins metabolite carrier precursor of IM
α-helical OM protein precursor
β-barrel OM protein precursor
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Current Opinion in Cell Biology
Overview of protein import and sorting pathways in mitochondria. More than 99% of mitochondrial proteins are nuclear encoded and synthesized as precursors on free ribosomes in the cytosol. Most of these precursors pass the outer mitochondrial membrane (OM) via the translocase of the outer membrane (TOM) complex. Precursors with cleavable N-terminal presequences are further sorted to the matrix or inner membrane (IM) by the translocase of the inner membrane (TIM23). Precursors of b-barrel proteins bind to small Tim chaperones in the intermembrane space (IMS) after passing the OM and are then sorted into the OM via the sorting and assembly machinery (SAM). Preproteins of the metabolite carrier family are translocated across the OM via TOM followed by small Tim-assisted transfer to the TIM22 translocase from which they get inserted into the IM. The MIA machinery (mitochondrial intermembrane space assembly) mediates import of intermembrane space proteins with characteristic cysteine motifs after their passage through the TOM complex. A few a-helical outer membrane proteins appear to be imported without the TOM complex but may instead require the MIM (mitochondrial import) machinery [22–27]. www.sciencedirect.com
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44 Cell regulation
(b) ALA synthase precursor
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DOWNREGULATION OF GLYOXYLATE SHUNT
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Malate Citric acid cylce
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HEME Succinyl-CoA + glycine Current Opinion in Cell Biology
Regulation of mitochondrial protein import by metabolites or activity of metabolic pathways. (a) Fumarase is dually distributed between the cytosol and mitochondria. The cytosolic pool of the enzyme takes part in the conversion of acetyl-CoA (C2) to C4 or C6 carbohydrates via the glyoxylate shunt. In mitochondria, fumarase is required for catabolic consumption of acetyl-CoA in the citric acid cycle. The fumarase precursor is imported via TOM and TIM23 machineries into the mitochondrial matrix where the N-terminal presequence is cleaved off (1). While a fraction of fumarase is completely imported into the mitochondrial matrix (2), another pool of the processed enzyme undergoes a fast folding during import that favors the retrograde translocation of the enzyme to the cytosol (3). The partitioning of fumarase between cytosol and mitochondria depends on the activity of the glyoxylate shunt. Its downregulation supports mitochondrial distribution of the enzyme. (b) 5-aminolevulinate (ALA) synthase is a mitochondrial enzyme that catalyzes the first step in heme biosynthesis. Nuclear-encoded ALA synthase precursor is synthetized in the cytosol and imported into the mitochondrial matrix via the TOM and TIM23 machinery. In a negative feed-back inhibition, binding of the end product heme to the N-terminal presequence of ALA synthase precursor impairs its import into the mitochondrial matrix.
an important role as a control station for metabolic pathways by feedback mechanisms upon changes in the concentration of metabolites. As the bulk of nuclear-encoded mitochondrial proteins initially pass through the TOM complex, regulation of the flux of mitochondrial proteins into the organelle at this stage can offer a direct means to adjust mitochondrial metabolism to cellular demands. Indeed, the TOM complex has emerged as a perfect communication hub for tuning mitochondrial activity. Its architecture, function and spatial distribution seem to be dynamically adjusted according to the metabolic condition [33,34,35,36]. Studies performed on Saccharomyces cerevisiae have unveiled molecular mechanisms of the regulation of the TOM machinery by distinct signalling circuits involving cytosolic and mitochondrial outer membrane bound protein kinases [33,34,35]. S. cerevisiae is an example of a facultative aerobic organism that is capable of rapid ATP production in a process called fermentation. Here, the end product of glycolysis, pyruvate, is reduced to ethanol and carbon dioxide. Oxygen and external glucose levels control the switch from respiration Current Opinion in Cell Biology 2015, 33:42–48
to fermentation in this organism. Shifting energy metabolism from OXPHOS to glycolysis is also characteristic to human muscle cells during extensive training and it is a hallmark of cancer cells (Warburg effect). Noteworthy, increasing evidence points out that such metabolic reprogramming is a prerequisite for tumor development . In S. cerevisiae, the TOM complex is targeted by kinases that are activated under fermentable conditions such as CK1 (casein kinase 1) and PKA (c-AMP-dependent kinase) [33,34,35] (Figure 3). Remarkably, although these kinases are induced by the same stimulus, they exert opposite effects on mitochondrial protein biogenesis reflecting the complexity of the signalling events that control mitochondrial import machineries. PKA is activated in response to enhanced glucose levels in a cascade that involves adenylyl cyclase (Cyr1) that converts ATP to cAMP. PKA activity is regulated by its regulatory subunit Bcy1, which in the absence of cAMP binds the catalytic subunits (Tpk1, Tpk2, and Tpk3) and inhibits kinase activity . Upon activation PKA phosphorylates precursors of the central constituents of the TOM complex in the cytosol and therefore acts directly in the regulation of the import machinery biogenesis itself. www.sciencedirect.com
Regulation of mitochondrial protein import machinery Opalin´ska and Meisinger 45
PKA (a) S76
Metabolite carrier precursor
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Metabolic signalling pathways regulate protein import at the mitochondrial entry gate TOM. In S. cerevisiae elevated glucose levels cause a metabolic switch towards glycolysis in conjunction with the downregulation of mitochondrial respiratory function. Glucose activates cytosolic PKA that in turn regulates the mitochondrial import machinery TOM at several levels: (a) PKA phosphorylates the precursors of Tom40 (serine 54) and Tom22 (threonine 76) in the cytosol. As a consequence, import and assembly of these central TOM subunits are impaired. (b) PKA can directly tune the flux of metabolite carrier precursors to mitochondria. PKA phosphorylates the import receptor Tom70 at serine 174 at mitochondria. This leads to an inhibition of the docking of metabolite carrier precursors bound to cytosolic chaperones (Hsp70). (c) Glucose induces translocation of CK1 to the plasma membrane (PM) and to the mitochondrial outer membrane (OM). Mitochondrial CK1 phosphorylates Tom22 (threonine 57) resulting in the stimulation of Tom22 assembly into the mature TOM complex (by enhanced interaction with Tom20). CK1 acts downstream of PKA and alleviates the negative impact of PKA on mitochondrial protein import.
Phosphorylation of the precursor of the channel forming subunit, Tom40 at position Ser54 and the core receptor Tom22 at residue Thr76 inhibits their import and negatively regulates TOM complex biogenesis [34,35]. Simultaneously, PKA can also directly modulate the flux of specific precursors through the TOM complex by affecting the receptor function of Tom70 . Tom70 facilitates the import of precursors of the metabolite carrier family that are located in the inner membrane such as ADP/ ATP carriers or phosphate carriers. These hydrophobic precursors are delivered to the mitochondrial surface with the assistance of cytosolic Hsp70 chaperones. Precursor loaded Hsp70s hereby dock at the TOM complex by interaction with Tom70. A negatively charged Glu-GluVal-Asp (EEVD) motif at the C-terminus of Hsp70 is critical for binding to the TOM complex . Upon a metabolic switch from respiratory to fermentative conditions, PKA phosphorylates the cytosolic domain of www.sciencedirect.com
Tom70 at position Ser174. The negative charges introduced by this phosphorylation event impair electrostatic interactions between Tom70 and precursor-bound Hsp70. As a consequence, the import of metabolite carriers, whose activity is not necessarily required under these metabolic conditions, is compromised . Although switching energy metabolism from respiration towards glycolysis requires down-regulation of mitochondrial respiratory functions, numerous life-sustaining metabolic reactions have to be maintained within the organelle. Hence, glucose also activates signalling circuits that exert stimulatory effects on the mitochondrial protein import machinery in order to mitigate negative effects of signal transduction cascades that results in attenuation of mitochondrial activity. Casein kinase 1 (CK1) is an example of a glucose-induced protein kinase alleviating the inhibitory effects of PKA. Current Opinion in Cell Biology 2015, 33:42–48
46 Cell regulation
CK1 is palmitoylated by the palmitoyl transferase Akr1 . This modification was thought to target the kinase mainly to the plasma membrane . However, a fraction of CK1 has been recently found at the outer mitochondrial membrane and this localization also depends on palmitoyl transferase activity . Mitochondrial localization of CK1 is promoted upon metabolic switch from respiratory to fermentative conditions. Mitochondrialocalized CK1 phosphorylates Tom22 at position Thr57 in the presence of active PKA. This phosphorylation supports the interaction of Tom22 with Tom20 and consequently promotes assembly of the TOM complex . Collectively, the coordinated action of diverse signalling circuits provides a means to accurately tune the function of the main entry gate for nuclear-encoded mitochondrial proteins according to metabolic requirements. The role of metabolic pathways is not limited to the supply of various building blocks and energy for cell life. Growing evidence suggests that metabolic pathways can directly influence signalling circuits and determine cell fate [14,15]. Mitochondrial function is strongly linked to cell division, growth and death. Casein kinase 2 (CK2) has a myriad of substrates that are involved in a variety of cellular functions vital for cell differentiation, proliferation and survival. CK2 is a highly conserved and constitutively active kinase [42– 44]. The central import receptor Tom22 has emerged as a CK2 target . CK2 does not only phosphorylate Tom22 precursor at Ser44 and Ser46 in the cytosol to promote its interaction with the import receptors, but also phosphorylates Tom22 at the mature TOM complex. The introduction of negative charges in this region of Tom22 stimulates its association with Tom20 and as a consequence maturation of the TOM complex . Similar to CK1, CK2 exerts a stimulatory role in mitochondrial biogenesis. A further target of CK2 that is involved in protein biogenesis represents Mim1. Mim1 is a core component of the MIM complex (Figure 1) that transiently interacts with the TOM complex  and that is involved in biogenesis of several TOM subunits including small Tom proteins, Tom20 and Tom70. Phosphorylation of Mim1 by CK2 at position Ser12 and Ser14 is critical for preserving sufficient levels of this protein at mitochondria  and thereby plays an important role in the biogenesis of the TOM complex. Taken together, the general protein entry gate in mitochondria is an important target for a complex network of overlapping signalling circuits that modulate not only protein import activity but also biogenesis of the import machinery. Tuning the flux of nuclear-encoded mitochondrial precursors to the organelle allows a rapid and careful balancing of mitochondrial activity and cellular metabolism.
Mitochondrial protein translocases emerged as final effectors of numerous interplaying signalling pathways. A growing number of studies support the notion that adjusting capacity and specificity of mitochondrial protein import machineries can provide an acute means of regulation of mitochondrial function [29,30,33,34,35,36,45,46,47]. Although detailed mechanisms that govern this phenomenon have been explored mostly in S. cerevisiae, it is very likely that they are applicable in higher eukaryotes as well. Yet, signalling pathways involved in regulation of mitochondrial import machineries and the consequences of their action might be divergent in various species. Moreover it is likely that such mechanisms are highly tissue-specific. Mitochondrial dysfunction is associated with a large number of human disorders, for example, type 2 diabetes, metabolic syndrome, obesity, neurodegenerative disorders, cancer or cardiomyopathies [2,48]. The view that mitochondrial homeostasis and function are substantially dependent on a direct adjustment of the activity of the mitochondrial protein import machinery opens a new and fascinating area of research that might contribute to our understanding of the etiology of many diseases and support their treatment. For instance, recent findings imply a role of import machinery components in cancer development: Numerous constituents of mitochondrial protein translocases are often upregulated in cancer tissues and this phenomenon is indicative of a poor survival prognosis [49–51,52,53,54]. Recently, the core component of the MIA machinery, Mia40, has been directly linked with metabolic adaptation required for cancerogenesis [52,55]. Furthermore, metabolic reprogramming during tumorigenesis often requires redistribution of kinases or transcription factors to mitochondria in a process that depends on the mitochondrial import machinery [55–58]. The molecular mechanisms behind these processes are currently unknown. Undoubtedly, the fascinating field of research underscored in this minireview, awaits further survey.
Acknowledgements We thank Dr. Nora Vo¨gtle for critically reading the manuscript. Our work is supported by the Deutsche Forschungsgemeinschaft and the Excellence Initiative of the German Federal & State Governments (EXC 294 BIOSS).
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