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Keywords:

  • CK2;
  • cAMP;
  • mitochondria;
  • protein import;
  • phosphorylation;
  • PKA;
  • phosphoproteomics;
  • S. cerevisiae ;
  • Tom22;
  • Tom70

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Uncovering the mitochondrial phosphoproteome
  5. Cytosolic kinases differentially regulate biogenesis of TOM precursor proteins
  6. CK2 regulates TOM biogenesis by phosphorylating key components involved in TOM complex assembly
  7. Metabolic switches: rapid adaption of TOM complex activity to cellular requirements
  8. Acknowledgements
  9. References

For decades, the pyruvate dehydrogenase complex in the mitochondrial matrix was considered as a rare example of how protein kinases and phosphatases can regulate important functions within this organelle. During the last decade, several proteomic studies revealed that a large fraction of mitochondrial proteins are indeed phosphorylated. A surprisingly high number of phosphorylation sites was found at the preprotein import machinery, TOM, in the outer membrane that provides the central protein import gate for most mitochondrial precursors synthesized in the cytosol. This review describes current knowledge of the mitochondrial phosphoproteome and introduces the first regulatory mechanisms of protein import dynamics by reversible phosphorylation, which have been uncovered mainly in the model organism Saccharomyces cerevisiae.


Abbreviations
CK2

casein kinase 2

PKA

protein kinase A

TOM

translocase of the outer membrane

Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Uncovering the mitochondrial phosphoproteome
  5. Cytosolic kinases differentially regulate biogenesis of TOM precursor proteins
  6. CK2 regulates TOM biogenesis by phosphorylating key components involved in TOM complex assembly
  7. Metabolic switches: rapid adaption of TOM complex activity to cellular requirements
  8. Acknowledgements
  9. References

Mitochondria are essential eukaryotic organelles and play a central role in energy metabolism via oxidative phosphorylation. Moreover, several mitochondrial biosynthetic pathways provide the cell with essential metabolites such as lipids, amino acids or iron-containing cofactors. In addition to being the powerhouse of the cell, mitochondria are integrated in multiple biological processes including cell-cycle control, cell differentiation, cell survival, programmed cell death, neuronal protection and aging [1-9].

Nevertheless, mitochondria have long been viewed as isolated and independent organelles that barely communicate with their environment. However, the diversity of mitochondrial functions suggests that the organelle's activities are not only restricted to mitochondria themselves, but mandatorily touch other cellular compartments. From this point of view, to integrate mitochondrial processes within the cellular network, mitochondria need to actively communicate with the rest of the cell.

The first evidence that a central mitochondrial metabolic function is regulated by reversible protein phosphorylation, probably the most generally used mechanism to regulate cellular processes [10], was reported in the late 1960s by Linn and colleagues [11]. They identified the E1 pyruvate dehydrogenase subunit of the pyruvate dehydrogenase complex as the first mitochondrial phosphoprotein by monitoring the incorporation of radiolabelled [32P]ATP into purified pyruvate dehydrogenase complex from beef kidney mitochondria [11-13].

During the last decade, an increasing number of mitochondrial phosphoproteins, protein kinases and phosphatases have been identified, indicating that reversible protein phosphorylation indeed plays a regulatory role in various mitochondrial functions, enabling the cell to integrate mitochondrial processes into the cellular network and facilitating a rapid response to changed cellular demands [1, 14-25]. Quite recently, it was demonstrated that the protein import machinery at the mitochondrial outer membrane in yeast (TOM complex) harbours more than 30 different phosphorylation sites. Furthermore, several cytosolic kinases were found to regulate biogenesis and the function of this central organelle's entry gate. This review summarizes our current understanding of how reversible protein phosphorylation regulates protein import into mitochondria, and how mitochondrial protein biogenesis is adjusted to changing requirements of the cell.

Uncovering the mitochondrial phosphoproteome

  1. Top of page
  2. Abstract
  3. Introduction
  4. Uncovering the mitochondrial phosphoproteome
  5. Cytosolic kinases differentially regulate biogenesis of TOM precursor proteins
  6. CK2 regulates TOM biogenesis by phosphorylating key components involved in TOM complex assembly
  7. Metabolic switches: rapid adaption of TOM complex activity to cellular requirements
  8. Acknowledgements
  9. References

To gain insights into mitochondrial functions and their integration into cellular signalling networks, it is important to know the protein composition of this organelle. Comprehensive proteomic studies have led to the identification of ~ 850 mitochondrial proteins in yeast and nearly 1100 proteins in mouse mitochondria, covering ~ 85% of the mitochondrial proteome in both cases. Among them were many signalling proteins including protein kinases, phosphatases, G proteins and GTPase-activating proteins, indicating the presence of signalling pathways, e.g. via reversible phosphorylation [18, 21-23, 26-29].

Initial global profiling of phosphoproteins in whole-cell extracts successfully identified several hundred (in yeast) to thousands (mammals) of phosphorylated proteins [30-34]. However, among them, only a very limited number of mitochondrial phosphoproteins could be detected.

The first systematic phosphoproteome studies using purified mitochondrial fractions increased the numbers of phosphoproteins identified because of increased sensitivity compared with total cell extracts. Reinders et al. [25] identified 80 phosphorylation sites in 48 different proteins in yeast mitochondria, whereas Lee et al. [35] found 84 phosphorylation sites within 62 different mitochondrial proteins in mouse. These novel mitochondrial phosphoproteins are not restricted to particular mitochondrial compartments, but are distributed all over the organelle. They are associated with various mitochondrial functions like oxidative phosphorylation, the tricarboxylic acid cycle, metabolite transport, lipid metabolism and genome maintenance.

The number of mitochondrial phosphoproteins has been growing continuously over recent years, indicating that reversible protein phosphorylation has been underestimated as a mechanism to modulate and integrate mitochondrial processes. Despite the growing number of identified mitochondrial phosphoproteins [36-44], their overall amount still seems to be far below the actual numbers. It has been estimated that 33% of cellular proteins will be phosphorylated at least once in their life cycle, suggesting that the currently known mitochondrial phosphoproteins represent only a small pool of the organelle's phosphoproteome [14, 45, 46]. A recent comprehensive proteomic study supports this assumption: Grimsrud et al. [47] applied multiplexed quantitative proteomics and site-specific phosphoproteomics to mouse liver mitochondria under distinct biological conditions. They identified 811 phosphosites on 295 mitochondrial proteins, indicating that reversible phosphorylation is a key mechanism regulating mitochondrial functions. Most likely, the yeast mitochondrial phosphoproteome known to date is far from being complete. This is also underlined by the fact that further subfractionation of mitochondria and analysis of purified native protein complexes in yeast, as demonstrated for the TOM complex, can lead to the identification of a multiple of phosphosites compared with enriched mitochondrial fractions [25, 48]. The TOM complex serves as the central protein entry point for most of the mitochondrial precursor proteins that have to be imported into the organelle from the cytosol. From here, several additional translocation machineries sort the incoming precursors into the four different mitochondrial compartments (Fig. 1A; see [49-51] for a detailed description of mitochondrial import pathways). With more than 30 different phosphorylation sites, the TOM complex holds the largest number of these modifications compared with other mitochondrial functional entities [31, 33, 36, 39, 40, 48, 52]. It is likely that phosphoproteomic profiling of other purified complexes, such as the respiratory complexes or the ribosome, will uncover a multitude of phosphorylation sites on these entities as well.

image

Figure 1. (A) Schematic overview of mitochondrial protein import pathways. Most precursors are translocated across the outer membrane (OM) via the TOM complex. Matrix-targeted preproteins with cleavable presequences, that possess positively charged amino acids are further sorted via the TIM23 complex in the inner membrane (IM). This process is driven by the membrane potential Δψ across the inner membrane and the import motor PAM in the matrix that hydrolyses ATP. Presequences can be cleaved by the mitochondrial processing peptidase (MPP). Depending on additional internal hydrophobic signals within the precursor, TIM23 can also sort precursors laterally into the IM. Import and oxidative folding of many intermembrane space (IMS)-destined proteins is mediated by the mitochondrial intermembrane space assembly (MIA) pathway that trap incoming precursors in the IMS by formation of disulfide bonds. The small TIM chaperones in the IMS escort precursors of the metabolite carrier family to the TIM22 complex which inserts these multispanning membrane proteins into the IM. Small TIM chaperones are also involved in the transfer of beta-barrel proteins from the TOM complex to the sorting and assembly machinery (SAM) in the OM, which mediates their membrane insertion and assembly. Certain proteins of the OM that contain α-helical transmembrane domains are not imported by the TOM complex but require the MIM complex [49-51, 91]. (B) Schematic representation of the TOM and MIM complex and overview of the function, topology and phosphorylation sites (P) of their single subunits.

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The TOM complex comprises seven different subunits: the TOM receptors Tom20, Tom22 and Tom70, the channel-forming Tom40 and three small subunits Tom5, Tom6 and Tom7. Each of these subunits is phosphorylated and most of the phosphorylation sites are located at the cytosolic side of the mitochondria, indicating a regulatory role for cytosolic protein kinases and phosphatases in protein import (Fig. 1B). The TOM complex therefore seems to be an ideal target for regulatory mechanisms that link mitochondrial protein biogenesis to cytosolic signalling networks.

Cytosolic kinases differentially regulate biogenesis of TOM precursor proteins

  1. Top of page
  2. Abstract
  3. Introduction
  4. Uncovering the mitochondrial phosphoproteome
  5. Cytosolic kinases differentially regulate biogenesis of TOM precursor proteins
  6. CK2 regulates TOM biogenesis by phosphorylating key components involved in TOM complex assembly
  7. Metabolic switches: rapid adaption of TOM complex activity to cellular requirements
  8. Acknowledgements
  9. References

In silico analysis combined with in vitro phosphorylation assays revealed that several cytosolic kinases are involved in the phosphorylation of TOM subunits. In particularly, casein kinase 2 (CK2) and protein kinase A (PKA) were identified as phosphorylating Tom proteins at various positions [18, 48, 53]. However, nothing was known about the functional relevance of these sites.

For Tom22, which has a dual role as an import receptor and multifunctional organizer of the TOM complex [54], residues Ser44 and Ser46 were shown to be quantitatively phosphorylated by CK2. Inactivation of CK2 as well as expression of a nonphosphorylatable Tom22S44,46A mutant resulted in decreased Tom22 protein levels in vivo. Interestingly, phosphorylation of Tom22S44,46 by CK2 occurs in the cytosol, before integration of the precursor into the outer membrane. The import of newly synthesized Tom22 precursor depends on pre-existing Tom receptors [50]. Nonphosphorylated Tom22 precursors showed impaired association with mitochondria, most likely due to less-efficient recognition of the precursor by Tom20. Consequently, CK2-dependent phosphorylation of Tom22S44,46 stimulated targeting to the mitochondrial outer membrane [48].

Protein phosphorylation is used as a mechanism to regulate protein localization within the cell [55]. Recently, it was reported that targeting of selected precursors to mammalian mitochondria is modulated by phosphorylation. In mammals, PKA in particular was identified as phosphorylating several precursor proteins, stimulating their interaction with cytosolic chaperones and thereby promoting their import into mitochondria [56-62]. By contrast, yeast PKA was found to inhibit import of the precursor of the protein translocation channel Tom40. Residue Ser54 was identified as a target of PKA, however, this position was never found to be phosphorylated at the mature TOM complex [48, 53]. The phosphorylated Tom40S54 precursor remains on the mitochondrial surface but is not able to interact with the Tom receptors, resulting in reduced membrane integration and assembly of Tom40 into the mature TOM complex (Fig. 2) [53].

image

Figure 2. Regulation of Tom40 import by PKA. Switching yeasts cells to fermentative growth conditions activates the catalytic PKA subunits (TPK) that phosphorylate Tom40 precursor in the cytosol at residue Ser54. As a consequence, import of phosphorylated Tom40 is blocked. Bcy1, regulatory subunit of yeast PKA.

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PKA is a serine/threonine kinase regulated by the second messenger cAMP. It plays a role in numerous cellular processes including organellar functions. PKA activity has been linked to mitochondrial functions, like respiration, morphology or apoptosis [1, 14, 56, 59, 60]. Inactive PKA forms a tetramer, consisting of two catalytic subunits, encoded in yeast by the TPK1, TPK2 and TPK3 genes and two regulatory subunits encoded by the single gene BCY1 [63-66]. Binding of cAMP to Bcy1 releases the active kinase [66-69]. In the facultative anaerobic organism Saccharomyces cerevisiae the cellular cAMP levels are regulated by the availability of fermentable carbon sources.

A switch of these nutrients, e.g. from respiratory carbon sources like glycerol to glucose or sucrose, is sensed by a plasma-membrane-located G-protein-coupled receptor, Gpr1 that activates adenylate cyclase Cyr1 via the G protein Gpa2. This immediately increases the cAMP levels, thereby activating PKA [67-70]. Such a metabolic switch from respiration to fermentation allows more rapid energy production in yeast cells [70]. Based on these metabolic changes, yeast cells no longer depend on ATP synthesis by mitochondrial oxidative phosphorylation, resulting in a decreased requirement for mitochondrial protein mass and therefore protein import. Impairment of Tom40 import by PKA seems to be a proper mechanism to reduce mitochondrial import rates under metabolic conditions that require less mitochondrial activity.

Taken together, CK2 and PKA can modulate the biogenesis of the TOM complex by phosphorylating Tom precursors in the cytosol. Their opposing actions in yeast allow adjustment of mitochondrial protein import which is required to adapt to changes in cellular metabolism.

CK2 regulates TOM biogenesis by phosphorylating key components involved in TOM complex assembly

  1. Top of page
  2. Abstract
  3. Introduction
  4. Uncovering the mitochondrial phosphoproteome
  5. Cytosolic kinases differentially regulate biogenesis of TOM precursor proteins
  6. CK2 regulates TOM biogenesis by phosphorylating key components involved in TOM complex assembly
  7. Metabolic switches: rapid adaption of TOM complex activity to cellular requirements
  8. Acknowledgements
  9. References

CK2 forms a tetramer consisting of two catalytic subunits, Cka1 and Cka2, as well as two regulatory subunits, Ckb1 and Ckb2 [71-74]. To date, the functional relevance of these regulatory subunits remains unclear [75, 76]. CK2 plays a role in many different cellular functions, like proliferation, gene expression or apoptosis, and several hundreds of targets have been identified. It is also involved in the regulation of chaperone activity and control of the circadian clock [71, 73-76]. Unlike most other protein kinases, CK2 is not activated by typical second messenger systems, but rather is constitutively active. However, the substrate specificity of CK2 seems to be regulated by different interaction partners, changes in conformation or cellular sublocalization [74, 76-79]. A high level of CK2 activity can be observed in fast-growing cells (both yeast and mammals) when increased protein biogenesis is required [77-79].

Tom22S44,46 phosphorylation by CK2 occurs in a constitutive way [48] and Tom22 precursor phosphorylation promotes targeting to mitochondria (see above and Ref. [48]). Beyond that, both phosphosites play an essential role in the interaction of the receptor protein Tom20 with the TOM complex [48]. Tom20 is the main import receptor that recognizes precursor proteins with an N-terminal presequence, which form the largest group of mitochondrial preproteins [49, 80]. Tom20 is only loosely attached to the TOM core complex, which consists of Tom40, Tom22 and the small Tom proteins Tom5, -6 and -7 [54, 81]. Tom22 serves as a docking site for Tom20, promoting the association between Tom20 and the TOM complex. This association of Tom20 and Tom22 is necessary for the efficient transfer of precursors from the receptor to the TOM core complex [18, 48]. The detailed structural organization of the TOM complex is not yet clear and little information is available about protein–protein interactions within the complex. Both phosphosites of Tom22, Ser44 and Ser46, are located within a region that strongly interacts with Tom20 [82]. Remarkably, Tom20 can be detached from the TOM complex upon phosphatase treatment of isolated mitochondria and this displacement is dependent on the Ser44 and Ser46 residues (Fig. 3) [48]. Therefore, phosphorylation also regulates the assembly of single components within the mature TOM complex.

image

Figure 3. CK2 controls biogenesis of the TOM complex. CK2-dependent phosphorylation of Tom22 at Ser44 and Ser46 stimulates targeting of Tom22 precursor and docking of the import receptor Tom20 via Tom22 at the TOM complex. Phosphorylation of Mim1 at Ser12 and Ser16 promotes stability of MIM, thereby facilitating import of Tom20 and Tom70 precursors.

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A further target of CK2 that is involved in assembly of the TOM complex is Mim1, a small outer membrane protein required for import of the receptors Tom20 and Tom70 [48, 83, 84]. Phosphorylation by CK2 at residues Ser14 and Ser16 is required to maintain stable Mim1 protein levels [18, 48]. As a consequence of low CK2 activity, Tom20 and Tom70 import is strongly impaired (Fig. 3) [48].

CK2 regulates the biogenesis of the TOM complex on three different levels: (a) targeting of the Tom22 precursor to the outer membrane, (b) promoting the association of Tom20 with the TOM complex, and (c) regulating Mim1-dependent import of the TOM receptors Tom20 and Tom70. Because of the interplay between these different steps, CK2 activity provides a powerful means to modulate assembly of the TOM complex (Fig. 3) and thereby control the import rates for cytosolic precursors into the organelle.

Regulated biogenesis of protein translocation machineries by reversible phosphorylation might be a common mechanism to modulate protein transport across cellular membranes. In particular, CK2 appears to also be involved in phosphorylating protein translocation machineries in other organelles, e.g. phosphorylation of Sec63, a member of the protein translocation complex in the endoplasmic reticulum in yeast [85]. Phosphorylation seems to stimulate formation of the translocase complex and to support efficient post-translational import into the endoplasmic reticulum [86]. Furthermore, protein import into chloroplasts might also be regulated by CK2. Toc159, a receptor subunit of the translocase of the outer chloroplast envelope, is phosphorylated at multiple sites by CK2. However, the physiological role of this process is not yet known [87]. In summary, CK2 might play a general role in regulating organelle biogenesis in cells by stimulating assembly of protein translocation systems.

Metabolic switches: rapid adaption of TOM complex activity to cellular requirements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Uncovering the mitochondrial phosphoproteome
  5. Cytosolic kinases differentially regulate biogenesis of TOM precursor proteins
  6. CK2 regulates TOM biogenesis by phosphorylating key components involved in TOM complex assembly
  7. Metabolic switches: rapid adaption of TOM complex activity to cellular requirements
  8. Acknowledgements
  9. References

The mechanisms described so far address the regulation of TOM complex biogenesis by cytosolic kinases. However, specific cellular changes might require a more rapid change in mitochondrial protein import rates. In such cases, a direct regulation of the import activity of the mature TOM complex would be more applicable. Moreover, these mechanisms could allow a selective change in the import rates of distinct classes of precursors and thereby modulate the various import routes into the mitochondria. Ideal targets for this regulation are the various Tom receptors that recognize and bind different classes of precursors [49-51].

Global phosphoproteomic studies of total yeast cell extract identified a phosphorylation site at residue Ser174 of the import receptor Tom70 [31, 36]. For these studies, the yeast cells had been grown under fermentative conditions. Interestingly, the Ser174 site could not be detected under respiratory conditions, either in highly purified outer mitochondrial membranes or in the isolated native TOM complex [48]. Indeed, it was demonstrated that Tom70S174 is specifically phosphorylated by PKA under fermentative conditions [48]. Tom70 is the major import receptor that recognizes precursors of the large family of metabolite carriers, like the ATP/ADP or phosphate carrier. In organello import of this precursor class was significantly impaired in a phosphomimetic mutant of Tom70, whereas import was enhanced when Ser174 was replaced by a non-phosphorylatable residue. After translation on cytosolic ribosomes, the hydrophobic precursors of the metabolite carriers are escorted to Tom70 by cytosolic chaperones of the Hsp70 family that can directly interact with the receptor [88]. Phosphorylation of Tom70S174 interferes with efficient binding of Hsp70 to Tom70 and consequently impairs import of the precursor (Fig. 4). Indeed, reduced protein levels of metabolite carriers can be observed in mitochondria upon in vivo activation of PKA [48].

image

Figure 4. Metabolic signal switch regulates function of the import receptor Tom70. Glucose-induced activation of PKA stimulates phosphorylation of Tom70 at Ser174 by the catalytic subunit TPK. This impairs interaction of Tom70 with cytosolic Hsp70 chaperones that deliver precursors of the metabolite carriers to the TOM complex.

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The cytosolic domain of Tom70 interacts via its chaperone-binding groove with the C-terminal EEVD motif of Hsp70 chaperones [88, 89]. Mutation of Arg171 in this chaperone-binding site disrupts binding of Hsp70 to Tom70 [88]. The PKA target Ser174 resides in close proximity to Arg171 and might introduce negative charges into the binding site. This, in turn, might influence the binding of the negatively charged EEVD chaperone motif and finally hinder interaction between receptor and chaperone. Thus phosphorylation of Tom70S174 by PKA directly regulates the function of this import receptor upon changes in metabolic conditions. This enables the yeast cell to adapt the import rate of mitochondrial metabolite carriers to an altered availability of nutrients.

It is likely that similar metabolic switches also exist in mammalian mitochondria and it will be interesting to identify the corresponding TOM targets and cytosolic kinase systems in higher organisms in future studies. It is possible that these switches also play a role in disease mechanisms, because some tumors can, for example, switch their metabolism from respiration to enhanced glycolysis rates, a phenomenon known as the Warburg effect [90]. Under these conditions, the exchange of metabolites might be downregulated by similar mechanisms as the import receptor of the metabolite carriers, Tom70, in yeast.

In summary, mitochondrial protein import has been shown to be a much more dynamic process, as expected. Recent work on the role of phosphorylation at the TOM complex revealed first insights into how mitochondrial protein biogenesis can adapt to various metabolic conditions (Fig. 5). It will be interesting to profile protein import machineries from inner mitochondrial compartments for phosphorylation. A major challenge will also be the identification of protein kinases and phosphatases that regulate mitochondrial function within the organelle. More than 50 years after the discovery of the pyruvate dehydrogenase kinase in the matrix the field awaits the identification of further resident mitochondrial kinases.

image

Figure 5. Schematic overview of cellular signalling systems that regulate mitochondrial protein import by phosphorylation at the TOM complex. CK2 seems to be activated by as yet unknown mechanisms under conditions when fast cell growth is required. Shifting of yeast cells from respiratory carbon sources to glucose induces a signalling cascade that involves activation of the G-protein-coupled receptor Gpr1 which activates the G protein Gpa2. Activation of the adenylate cyclase Cyr1 by Gpa2 leads to increased cAMP level. cAMP binds to the regulatory components of yeast PKA, Bcy1, thereby activating the catalytic subunits TPK.

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Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Uncovering the mitochondrial phosphoproteome
  5. Cytosolic kinases differentially regulate biogenesis of TOM precursor proteins
  6. CK2 regulates TOM biogenesis by phosphorylating key components involved in TOM complex assembly
  7. Metabolic switches: rapid adaption of TOM complex activity to cellular requirements
  8. Acknowledgements
  9. References

We thank Dr N. Vögtle for critically reading of the manuscript. Our work was supported by the Deutsche Forschungsgemeinschaft, Excellence Initiative of the German Federal & State Governments (EXC 294 BIOSS) Bundesministerium für Bildung und Forschung (Dynamo), and Trinationales Graduiertenkolleg GRK 1478.

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  2. Abstract
  3. Introduction
  4. Uncovering the mitochondrial phosphoproteome
  5. Cytosolic kinases differentially regulate biogenesis of TOM precursor proteins
  6. CK2 regulates TOM biogenesis by phosphorylating key components involved in TOM complex assembly
  7. Metabolic switches: rapid adaption of TOM complex activity to cellular requirements
  8. Acknowledgements
  9. References
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